Device, kit and method for pulsing biological samples with an agent and stabilising the sample so pulsed

ABSTRACT

A device and kit for pulsing a biological sample with a pulsing agent is disclosed. The biological sample so pulsed is subsequently stabilized and the device or kit provides a control reaction. Applications for the described device and kit are found in the field of medical diagnostics, particularly immunology.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.12/253,539, filed Oct. 17, 2008, now U.S. Pat. No. 7,947,450, issued May24, 2011, which is a continuation-in-part of U.S. application Ser. No.10/563,503, filed Mar. 2, 2006, now U.S. Pat. No. 7,700,276, issued Apr.20, 2010, both of which are incorporated herein by reference, which isthe U.S. National Phase under 35 U.S.C. §371 of InternationalApplication PCT/EP2003/007453, filed Jul. 10, 2003.

FIELD OF THE INVENTION

The present invention relates to devices, method and kits for use indiagnostic assays, and has applications in the field of immunology.

Introduction

Monitoring nucleic acid levels, for example, those of mRNA is valuablein ascertaining directly the effect of an agent on a biological system.For example, if an agent is introduced into a biological system for adefined length of time, the reaction of the system to the agent can bedetermined by measuring the levels of mRNA. This may be useful inmonitoring immunity wherein, for example, the agent is an antigen andthe mRNA monitored is cytokine mRNA e.g. interleukins.

Testing the impact of an agent by withdrawing blood from an individualand adding the agent at a later point in time introduces a variabledelay between the blood being out of circulation and stimulation withthe agent. During the delay, the blood may undergo slow or fast chemicalmodification, depending, for example, on the temperature at which it isheld. Furthermore, a delay which is variable means comparative studiesbetween consecutively withdrawn samples are invalid.

When testing for nucleic acid, a major challenge is due to theinstability of RNA in vitro especially when there is a requirement forthe detection of low-level RNA or unstable RNA. Even the degradation ofonly a small fraction of the RNA may change the interpretation of thelevels of RNA. Some transcripts are known to be present at low copy in acell; other transcripts have an “AU-rich” sequence in their 3′ endpromoting their fast degradation by endogenous RNAses. Studies haveshown that RNA rapidly degrades significantly within hours after samplecollection. Furthermore, certain species of RNA, through the process ofgene induction, increase once the sample is collected. Both RNAdegradation and in vitro gene induction can lead to an under- orover-estimation of the in vivo gene transcript number.

When measuring the effect of an agent on withdrawn blood, therefore, achallenge in the art is to manage the process of mRNA degradation whichbegins immediately after the blood is withdrawn and resumes after theintroduction of antigen. Because the ‘before’ degradation can have animpact on the ‘after’ degradation, the error is coupled to twoprocesses; hence the potential for error is greater and the error ismore difficult to delineate.

Another problem in the art is the necessity for several pieces ofequipment in performing an exposure of a biological sample to an agentfollowed by a subsequent nucleic acid analysis thereof. Typically,reagent bottles, accurate pipettors, refrigeration means are at leastrequired to perform quantitative measurements. If samples are beingtaken in the absence of suitable laboratory facilities, for example inthe home of an individual or in a basically-equipped surgery, it may notbe suitable or convenient to perform accurate substrate additions, andfurthermore refrigeration facilities might not be available.

Once a sample has been exposed to an agent, many methods exist toisolate and measure nucleic acids therein, for example mRNA. Somemethods allow even the determination of low-level transcripts out of apool of transcripts. However, none of them provide the possibility todetermine the level(s) of transcript(s) present in the biological sampleat the time of the sampling. Even under refrigerated conditions, thestorage of biological samples leads to incorrect mRNA levels. Indeed, inpractice, the analysis of fresh sample is not feasible as the place ofsampling and the place of RNA analysis are located differently.

Recently, PreAnalytiX (a joint venture between Becton Dickinson andQiagen) produced the PAXgene™ Blood RNA System. The PAXgene™ Blood RNASystem (also referred to as the Qiagen method) is an integrated andstandardized system for the collection and stabilization of whole bloodspecimens and isolation of cellular RNA. According to PreAnalytiX, inthe PAXgene™ Blood RNA System, blood is collected directly into PAXgene™Blood RNA Tubes and RNA is subsequently isolated using the PAXgene™Blood RNA Kit. Using this system, intact cellular RNA can be retrievedfrom whole blood.

The PAXgene™ Blood RNA Tube is a plastic, evacuated tube, for thecollection of whole blood and stabilization of the cellular RNA profile.The tubes contain an additive (a proprietary blend of reagents) thatstabilizes cellular RNA and may eliminate ex vivo induction of genetranscription and prevents the drastic changes in the cellular RNAexpression profiles that normally take place in vitro. RNA is thenisolated using silica-gel-membrane technology supplied in the PAXgene™Blood RNA Kit. According to PreAnalytiX, the resulting RNA accuratelyrepresents the expression profile in vivo and is suitable for use in arange of downstream applications. According to the supplier, accuratequantification of gene transcripts is possible using this system. Amajor disadvantage of this PAXgene™ Blood RNA System is that respectivePAXgene™ Blood RNA Tube needs to be combined with the PAXgene™ Blood RNAKit (see instruction manual of the PAXgene™ Blood RNA Tubes). Thisobliged combination, however, limits further improvement of the system.

More recently, the Tempus™ Blood RNA Tube (Applied Biosystems) has beendeveloped which is similarly is a plastic, evacuated tube, for thecollection of whole blood and stabilization of the cellular RNA profile.Each tube contains an additive (a proprietary blend of reagents) thatstabilizes cellular RNA and may eliminate ex vivo induction of genetranscription and prevents the drastic changes in the cellular RNAexpression profiles that normally take place in vitro.

Aims of the Invention

One aim of the present invention is to provide a device, kit and methodfor exposing a biological sample to an agent and stabilising the nucleicacid in the sample so exposed.

One aim of the present invention is to provide a device, kit and methodfor exposing a biological sample to an agent, stabilising the nucleicacid in the sample so exposed, which device, kit and method also providea control reaction.

Another aim of the present invention is to provide a device, kit andmethod which reduces, makes constant, or makes nearly constant the timebetween obtaining the biological sample and exposing said sample to anagent.

Another aim of the present invention is to provide a device, kit andmethod which reduces, makes constant or makes nearly constant the timefrom which the sample is exposed to an agent and the nucleic acid insaid sample is stabilised.

Another aim of the present invention is to provide a device, kit andmethod which exposes an agent to a sample without the need to measure anamount of sample and/or agent.

Another aim of the present invention is to provide a single device,which exposes an agent and a control to a sample, and makes constant ormakes nearly constant the time from which the sample and control areexposed to an agent and the nucleic acid in said sample is stabilised.

Another aim of the present invention is to provide a device, kit andmethod which exposes an agent to a sample without the need to measure anamount of stabilising agent.

Another aim of the present invention is to provide a device, kit andmethod for exposing a biological sample to an agent, stabilising thenucleic acid in the sample so exposed and extracting the nucleic acidtherefrom for further analysis.

Another aim of the present invention is to provide a device for exposinga biological sample to an agent, stabilising the nucleic acid in thesample so exposed, which device also provide a control reaction andfacilitates robotic automation.

Another aim of the present invention is provide a device, kit and methodwhich addresses a combination of one or more of the aforementioned aims.

SUMMARY OF THE INVENTION

One embodiment of the invention is a kit for assaying a liquidbiological sample comprising:

a vessel suitable for accepting liquid biological sample, exposing saidsample to a first substance and subsequently a nucleic acid stabilisingagent, said vessel comprising:

a) a first substance present inside said vessel,

b) a container in which said stabilising agent is present,

c) a connection between the inside of said vessel and the inside of saidcontainer,

d) a physical barrier that temporarily blocks said connection;

and

a control vessel suitable for accepting liquid biological sample,exposing said sample to a control substance and subsequently a nucleicacid stabilising agent, said control vessel comprising:

a) a control substance present inside said control vessel,

b) a control container in which said stabilising agent is present,

c) a connection between the inside of said control vessel and the insideof said control container,

d) a physical barrier that temporarily blocks said connection.

Another embodiment of the invention is a kit as described above, whereinsaid first substance is immobilised on part or all of the inside surfaceof said vessel.

Another embodiment of the invention is a kit as described above, whereinsaid first substance is immobilised on a solid support.

Another embodiment of the invention is a kit as described above, whereinsaid first substance is a liquid.

Another embodiment of the invention is a kit as described above, whereinsaid first substance is a solid.

Another embodiment of the invention is a kit as described above, whereinthe vessel and/or control vessel comprise one or more areas suitable forpuncture by a syringe needle.

Another embodiment of the invention is a kit as described above, whereinsaid area is a re-sealable septum.

Another embodiment of the invention is a kit as described above, whereinthe vessel and/or control vessel comprise a fitting suitable forreceiving a syringe and transmitting the contents therein to theinterior of said vessel or control vessel.

Another embodiment of the invention is a kit as described above, whereinthe vessel and/or control vessel comprise a fitting suitable forreceiving a syringe needle.

Another embodiment of the invention is a kit as described above, whereinthe vessel and/or control vessel comprise a valve which is capable ofminimising the flow of gas/liquid from vessel, and allowing the flow ofliquid biological sample into the vessel.

Another embodiment of the invention is a kit as described above, whereinthe vessel and/or control vessel comprise a means through whichdisplaced gas may be expelled.

Another embodiment of the invention is a kit as described above, whereinthe vessel and/or control vessel comprise is held under negativepressure.

Another embodiment of the invention is a kit as described above, whereinthe physical barrier of item d) is opened by the application of physicalforce to said vessel or control vessel.

Another embodiment of the invention is a kit as described above, whereinsaid force transmits an opening means to said physical barrier.

Another embodiment of the invention is a kit as described above, whereinsaid force irreversibly opens said physical barrier.

Another embodiment of the invention is a kit as described above, whereinsaid vessel and/or control comprises an indication for dispensing aknown volume of stabilising agent therein.

Another embodiment of the invention is a kit as described above, whereinsaid first substance comprises one or more immune system antigens.

Another embodiment of the invention is a kit as described above, whereinsaid immune system antigens are vaccine components.

Another embodiment of the invention is a kit as described above, whereinsaid immune system antigens are antigens which provoke a hyperallergenicresponse.

Another embodiment of the invention is a kit as described above, whereinsaid immune system antigens are one or more selected fromhistocompatibility antigens, bacterial LPS, tetanous toxoid, a cancerimmunotherapy antigen, MAGE-3, a cat allergen, Feld1, antigen presentingcells from an organ donor, an autoantigen, GAD65.

Another embodiment of the invention is a kit as described above, whereinsaid stabilising agent is an inhibitor of cellular RNA degradationand/or gene induction.

Another embodiment of the invention is a kit as described above, whereinsaid inhibitor of cellular RNA degradation and/or gene induction is thatas found in a PAXgene™ or Tempus™ Blood RNA Tube.

Another embodiment of the invention is a kit as described above, whereinthe exterior of the vessel and control vessel are joined to form asingle entity.

Another embodiment of the invention is an assay device for a liquidbiological sample, which facilitates exposure of said sample separatelyto a first substance and a control substance, and subsequently to anucleic acid stabilizing agent, the device comprising:

-   -   a first compartment in which said first substance is present,    -   a second compartment in which said control substance is present,    -   a third compartment where said stabilizing agent is present, and    -   a support for a biological sample tube.

Another embodiment of the invention is an assay device as describedabove, wherein one or more of the compartments is sealed, and comprisesone or more areas suitable for puncture by a hollow needle.

Another embodiment of the invention is an assay device as describedabove, wherein said area is a re-sealable septum.

Another embodiment of the invention is an assay device as describedabove, further comprising a support for demountable attachment to atransfer tubing, which transfer tubing comprises a hollow flexible orrigid tubing disposed with a hollow needle at either end, adapted fortransfer of liquid between any two compartments, or between acompartment and the biological sample tube.

Another embodiment of the invention is an assay device as describedabove, further comprising the transfer tubing as defined above.

Another embodiment of the invention is an assay device as describedabove, wherein one needle of the transfer tubing is attached in parallelalignment to the hollow needle of a pressurising tubing, whichpressurising tubing comprises a flexible hollow tubing disposed at oneend with said needle, and is adapted to apply vacuum or pressure to acompartment or biological sample tube to propel liquid through thetransfer tubing.

Another embodiment of the invention is an assay device as describedabove, wherein said first substance is immobilised on part or all of theinside surface of said vessel.

Another embodiment of the invention is an assay device as describedabove, wherein one or more of the compartments comprises an air vent.

Another embodiment of the invention is an assay device as describedabove, wherein the first and/or second compartments are held undernegative pressure.

Another embodiment of the invention is an assay device as describedabove, wherein the first and/or second compartments comprise anindication for dispensing a known volume of stabilising agent therein.

Another embodiment of the invention is an assay device as describedabove, wherein said first substance is as defined above.

Another embodiment of the invention is an assay device as describedabove, wherein said stabilizing agent is as defined above.

One embodiment of the present invention is a vessel suitable foraccepting a liquid biological sample, exposing said sample to a firstsubstance and subsequently a nucleic acid stabilising agent, said vesselcomprising:

a) a first substance present inside said vessel,

b) a container in which said stabilising agent is present,

c) a connection between the inside of said vessel and the inside of saidcontainer,

d) a physical barrier that temporarily blocks said connection. Anotherembodiment of the present invention is a vessel as described abovewherein said first substance is immobilised on part or all of the insidesurface of said vessel. Another embodiment of the present invention is avessel as described above wherein said first substance is immobilised ona solid support. Another embodiment of the present invention is a vesselas described above wherein said first substance is a liquid. Anotherembodiment of the present invention is a vessel as described abovewherein said first substance is a solid. Another embodiment of thepresent invention is a vessel as described above comprising one or moreareas suitable for puncture by a syringe needle. Another embodiment ofthe present invention is a vessel as described above wherein said areais a re-sealable septum. Another embodiment of the present invention isa vessel as described above comprising a fitting suitable for receivinga syringe and transmitting the contents therein to the interior of saidvessel. Another embodiment of the present invention is a vessel asdescribed above comprising a fitting suitable for receiving a syringeneedle. Another embodiment of the present invention is a vessel asdescribed above comprising a cannular suitable for withdrawing bodilyfluids. Another embodiment of the present invention is a vessel asdescribed above comprising a valve which is capable of minimising theflow of gas/liquid from vessel, and allowing the flow of liquidbiological sample into the vessel. Another embodiment of the presentinvention is a vessel as described above comprising a means throughwhich displaced gas may be expelled. Another embodiment of the presentinvention is a vessel as described above wherein said vessel is heldunder negative pressure. Another embodiment of the present invention isa vessel as described above wherein the physical barrier of item d) isopened by the application of physical force to said vessel. Anotherembodiment of the present invention is a vessel as described abovewherein said force transmits an opening means to said physical barrier.Another embodiment of the present invention is a vessel as describedabove wherein said force irreversibly opens said physical barrier.Another embodiment of the present invention is a vessel as describedabove wherein said vessel comprises an indication for dispensing a knownvolume of stabilising agent therein. Another embodiment of the presentinvention is a vessel as described above wherein said first substancecomprises one or more immune system antigens. Another embodiment of thepresent invention is a vessel as described above wherein said immunesystem antigens are vaccine components. Another embodiment of thepresent invention is a vessel as described above wherein said immunesystem antigens are antigens which provoke a hyperallergenic response.Another embodiment of the present invention is a vessel as describedabove wherein said immune system antigens are one or more selected fromhistocompatibility antigens, bacterial LPS, tetanous toxoid, a cancerimmunotherapy antigen, MAGE-3, a cat allergen, Feld1, antigen presentingcells from an organ donor, an autoantigen, GAD65. Another embodiment ofthe present invention is a vessel as described above wherein saidstabilising agent is an inhibitor of cellular RNA degradation and/orgene induction. Another embodiment of the present invention is a vesselas described above wherein said inhibitor of cellular RNA degradationand/or gene induction is that as found in a PAXgene™ or Tempus™ BloodRNA Tube. Another embodiment of the present invention is a method ofpulsing a sample of blood with an antigen, subsequently inhibitingcellular RNA degradation and/or gene induction therein and subsequentlytesting RNA components in the stabilised blood sample so pulsedcomprising the use of a vessel as described above.

Another embodiment of the present invention is a method of testing theimmune response of an individual towards an antigen comprising the useof a vessel as described above wherein the first substance is theantigen under investigation, comprising the steps of:

a) introducing a sample of blood taken from said individual into thevessel,

b) optionally agitating said vessel,

c) introducing after a pre-determined period of time, said nucleic acidstabilising agent into said vessel, and

d) testing the levels of mRNA.

Another embodiment of the present invention is a method as describedabove where step d) further comprises the steps of

e) forming a precipitate comprising nucleic acids,

f) separating said precipitate of step (e) from the supernatant,

g) dissolving said precipitate of step (f) using a buffer, forming asuspension,

h) isolating nucleic acids from said suspension of step (g) using anautomated device,

i) dispersing/distributing a reagent mix for RT-PCR using an automateddevice,

j) dispersing/distributing the nucleic acids isolated in step (h) withinthe dispersed reagent mix of step (i) using an automated device, and,

k) determining the in vivo levels of transcripts using the nucleicacid/RT-PCR reagent mix of step (j) in an automated setup.

Another embodiment of the present invention is a method as describedabove wherein the immune response of an individual towards an antigenagainst which the individual has been pre-immunised is tested, the firstsubstance is the antigen under investigation and the levels of cytokinemRNA are tested. Another embodiment of the present invention is a methodas described above wherein said cytokine is one or more of IL-2, IL-4,IL-13, IFN-gamma. Another embodiment of the present invention is amethod as described above wherein the hyperallergenicity of anindividual towards an antigen is tested, the first substance is theantigen under investigation and the levels of IL-4 mRNA are tested.Another embodiment of the present invention is a method as describedabove wherein the rejection of an organ transplant in an individualtowards an antigen is tested, wherein the first substance is ahistocompatibility antigen of the donor and the levels of IL-2 mRNA aretested. Another embodiment of the present invention is a use of a vesselas described above for pulsing a sample of blood with an antigen,subsequently inhibiting cellular RNA degradation and/or gene inductiontherein and subsequently testing RNA components in the stabilised bloodsample so pulsed. Another embodiment of the present invention is a useof a vessel as described above for extracting a pre-determined volumesample of blood from an individual using said needle or cannular,pulsing said sample with an antigen, subsequently inhibiting cellularRNA degradation and/or gene induction therein and subsequently testingRNA components in the stabilised blood sample so pulsed.

Another embodiment of the present invention is a kit suitable forpulsing a liquid biological sample with a first substance, andsubsequently introducing an agent that inhibits cellular RNA degradationand/or gene induction thereto, and testing mRNA components in thestabilised blood sample so pulsed, said kit comprising:

a) a vessel in which said first substance is present, and

b) a container in which said agent is present. Another embodiment of thepresent invention is a kit as described above wherein the inside of saidvessel and the inside of said container are connected, and a physicalbarrier temporarily blocks said connection. Another embodiment of thepresent invention is a kit as described above wherein said firstsubstance is immobilised on part or all of the inside surface of saidvessel. Another embodiment of the present invention is a kit asdescribed above wherein said first substance is immobilised on a solidsupport. Another embodiment of the present invention is a kit asdescribed above wherein said first substance is a liquid. Anotherembodiment of the present invention is a kit as described above whereinsaid first substance is a solid.

Another embodiment of the present invention is a kit as described abovewherein said vessel comprises one or more openings. Another embodimentof the present invention is a kit as described above said vesselcomprises one or more areas suitable for puncture by a syringe needle.Another embodiment of the present invention is a kit as described abovewherein said area is a re-sealable septum. Another embodiment of thepresent invention is a kit as described above wherein said vesselcomprises one or more fittings suitable for receiving a syringe andtransmitting the contents therein to the interior of said vessel.Another embodiment of the present invention is a kit as described abovewherein said vessel comprises one or more fittings suitable forreceiving a hypodermic syringe needle. Another embodiment of the presentinvention is a kit as described above wherein said vessel comprises oneor more cannulars suitable for withdrawing bodily fluids. Anotherembodiment of the present invention is a kit as described above whereinsaid vessel comprises one or more valves which are capable of minimisingthe flow of liquid from vessel, minimising the flow of gas into or fromvessel, and/or allowing the flow of liquid biological sample into thevessel. Another embodiment of the present invention is a kit asdescribed above wherein said vessel comprises one or more means throughwhich displaced gas may be expelled. Another embodiment of the presentinvention is a kit as described above wherein said vessel is held undernegative pressure. Another embodiment of the present invention is a kitas described above wherein the physical barrier of item d) is opened bythe application of physical force to said vessel. Another embodiment ofthe present invention is a kit as described above wherein said forcetransmits an opening means to said physical barrier. Another embodimentof the present invention is a kit as described above wherein said forceirreversibly opens said physical barrier. Another embodiment of thepresent invention is a kit as described above wherein said vessel and/orcontainer comprises an indication for dispensing a known volume ofstabilising agent therein. Another embodiment of the present inventionis a kit as described above wherein said first substance comprises oneor more immune system antigens. Another embodiment of the presentinvention is a kit as described above wherein said immune systemantigens are vaccine components. Another embodiment of the presentinvention is a kit as described above wherein said immune systemantigens are antigens which provokes a hyperallergenic response. Anotherembodiment of the present invention is a kit as described above whereinsaid immune system antigens are are selected from one or more ofhistocompatibility antigens, bacterial LPS, tetanous toxoid, a cancerimmunotherapy antigen, MAGE-3, a cat allergen, Feld1, antigen presentingcells from an organ donor, an autoantigen, and GAD65. Another embodimentof the present invention is a kit as described above wherein saidinhibitor of cellular RNA degradation and/or gene induction is that asfound in a PAXgene™ Blood RNA Tube. Another embodiment of the presentinvention is a kit as described above for testing the immune response ofan individual towards an antigen against which the individual has beenpre-immunised wherein the first substance is the antigen underinvestigation and the mRNA tested is cytokine mRNA. Another embodimentof the present invention is a kit as described above wherein saidcytokine is one or more of IL-2, IL-4, IL-13, IFN-gamma. Anotherembodiment of the present invention is a kit as described above fortesting an individual for hyperallergenicity towards an antigen whereinthe first substance is the antigen under investigation and the mRNAtested is IL-4 mRNA. Another embodiment of the present invention is akit as described above for testing an individual for rejection of anorgan transplant wherein the first substance is a histocompatibilityantigen of the donor and mRNA tested is IL-2 mRNA. Another embodiment ofthe present invention is a kit as described above further comprising oneor more oligonucleotides suitable for said testing said mRNA(s).

DETAILED DESCRIPTION OF THE INVENTION

One aspect of the present invention is related to a vessel suitable forholding a biological sample, said vessel holding a predetermined amountof a first substance which may be a pulsing agent.

As used herein “pulsing agent” comprises any substance to which abiological sample may be exposed. Examples of substances includepeptides, nucleic acids, antigens. The pulsing agent may comprise othercomponents besides the substance, such as stabilising agents,indicators, linkers, matrices, etc.

The term “biological sample” means a sample containing nucleicacids/biological agents such as clinical (e.g. cell fractions, wholeblood, plasma, serum, urine, tissue, cells, etc.), agricultural,environmental (e.g. soil, mud, minerals, water, air), food (any foodmaterial), forensic or other possible samples. With ‘whole blood’ ismeant blood such as it is collected by venous sampling, i.e. containingwhite and red cells, platelets, plasma and eventually infectious agents;the infectious agents may be viral, bacterial or parasitical. Theclinical samples may be from human or animal origin. The sample analysedcan be both solid or liquid in nature. It is evident when solidmaterials are used, these are first dissolved in a suitable solution,which could be the RNAlater reagent sold by Qiagen. According to theinvention, this solution is not always a real “buffer” with at least twowell balanced components. It may be a strong hypotonic solution such asNaCl alone or an extraction solution such as with alcohol.

The vessel, also known herein as a reaction vessel, may hold the pulsingagent in several ways. According to one aspect of the invention, thepulsing agent may be immobilised on the inside wall of the vessel. Theinside wall of the vessel may be lined with a suitable coating enablingthe pulsing agent to be attached. Alternatively, the pulsing agent maybe attached directly to part or all of the inside wall of the vessel.Suitable coatings, methods and vessel materials for suitable for suchattachments are known in the art. According to another aspect of theinvention the pulsing agent is present as a solid. The solid may be apowder, a freeze-dried pellet, a gel, a cream. Suitable solidcompositions and method of their preparation are known in the art.According to another aspect of the invention the pulsing agent isimmobilised on a solid support. The solid support may be attached to theinside of the vessel. Alternatively the solid support may be free of theinside of the vessel. Examples of solid supports include, but are notlimited to, chromatography matrix, magnetic beads. According to anotheraspect of the present invention, pulsing agent is present as a liquid.Suitable liquid compositions and method of their preparation are knownin the art.

Performing an analytical pulsing experiment outside of laboratoryconditions requires calibrated measuring equipment such as pipettors.Errors due to uncalibrated measuring devices can lead to inherent errorsand also human error in dispensing can lead to error between differentsamples, making comparative analysis invalid. Providing a vesselsupplied with a pre-determined amount of pulsing agent obviates the needfor additional equipment and eliminates human measuring errors.Moreover, the provision of a separate control vessel allowsnormalisation of results, and facilitates the detection of errors. Theuse of a control is important since RNA is labile and rapidly degradesonce it is withdrawn from the circulation. Inconsistent delays inpulsing whole blood would lead to un-comparable results. Duringincubation with a pulsing agent, depending on the agent; different(biological) pathways are engaged. The present invention allowsmeasurement of such a biological process by quantifying the messengerRNA of biomarkers such as cytokines, chemokines, transcription factorsand other gene products or surrogate markers representative for aspecific process. Without a reference value, the quantification of thesebiomarkers in the assay has little meaning; the use of an integratedcontrol permits rapid execution of a control reaction. By comparingassay and control, the meaning of the quantity of the biomarker and theinfluence of the pulsing agent on the biological system. As an example,it is general accepted that the Mx1 gene is under the control of IFNtype I. We took advantage of this knowledge and used this gene productas a surrogate marker to measure the therapeutic efficacy of IFN type I.The Mx1 gene product is quantified after incubating whole blood with IFNtype I. The quantity of Mx1 gene product can be expressed as a number ofmRNA copies compared to a number of mRNA copies of a normalization geneproduct, i.e. a gene whose transcription activity is not influenced bythe action of IFN. But this quantification would be meaningless withouta control reaction being a quantification of the surrogate marker undernon-stimulated control conditions i.e. same treatment without IFN typeI. The same reasoning can be made for any immune reaction. In order tohave a strict control, it is of great importance that the control sampleis treated the same way, i.e. is from the same withdrawal, sameincubation time and temperature and parallel treatment; the combinationof assay and control compartments in a single device makes thisfeasible.

Types of vessel according to the invention can be any suitable forstorage of biological samples. According to one aspect of the invention,the vessel containing the pulsing agent is sealed. According to oneaspect of the invention, the vessel containing the pulsing agent has aresealing means such as a screw-cap, push-on cap, a flip-cap. See, forexample, FIG. 5. According to one aspect of the invention, thebiological sample or other fluids may be introduced into the vessel bypuncture, using a syringe needle into the wall of the vessel. The wallof the vessel may be resealable after puncture, or the wall of thevessel may not be resealable after puncture, or the wall of the vesselmay be provided with a resealable area such as a septum. See, forexample, FIG. 4.

According to one aspect of the invention, the biological sample, orother liquids are introduced by means of a transfer tubing comprising ahollow tube disposed with a needle either end (e.g. a cannular needle).One needle is configured to puncture a septum of a sample tube holdingthe sample, or other container, and the other needle is configured topuncture the septum of the vessel; biological sample is able to betransferred from the sample tube to the vessel using the needle. Thetubing may be flexible or rigid. The shape of the transfer tubing may beU-shaped. See, for example, FIG. 22.

According to one aspect of the invention, the biological sample may beintroduced into the vessel by means of one or more fittings attached tovessel for receiving a syringe or other container fitted with a couplingmeans. For example, the vessel might be fitted with a Luer fitting thatcan receive a needleless syringe. See for example, FIG. 3. In anotherexample, the vessel might be fitted with a non-Luer fitting which canmate with a container having a reciprocating non-Luer design ofcoupling.

According to one aspect of the invention, the biological sample may beintroduced into the vessel by means of a cannular or hypodermic needlefitted to said vessel, suitable for directly withdrawing biologicalsamples from an individual. See, for example, FIG. 6.

According to one aspect of the invention, the biological sample may beintroduced into the vessel by opening the resealing means. See, forexample, FIG. 5.

As known by the skilled person, the introduction of a sample into asealed vessel will result in the displacement of an equal volume of airor gas therefrom, or a build up of pressure therein. Therefore, thevessel may be provided with a suitable means to allow displaced gas toexit from said vessel, or to accommodate the build up of pressure. Saidmeans are known the art and include valves, non-drip holes, vents,clothed-vents, expandable vessel walls, use of negative pressure withinsaid vessel. See, for example, arrow 31 on FIG. 11.

In one aspect of the invention the pressure inside the sealed vessel isnegative. The negative pressure may be utilised to relieve the pressurebuild-up upon introduction of biological sample into said sealed vessel.Alternatively, or in addition, the negative pressure may be at apredetermined level and may be utilised so as to allow the introductionof a fixed volume of biological sample.

According to another aspect of the invention, the vessel is providedwith a sealable septum as described above, adapted to receive a firstneedle (cannular) through which liquid biological sample can enter thevessel and a second needle through which positive or negative pressurecan be applied to the inside of the vessel. When negative (vacuum)pressure is applied through the second needle, biological sample can bedrawn into the vessel through the first needle. Preferably the firstneedle is part of the transfer tubing described earlier. The secondneedle may be part of a pressurizing tubing described below, which isconnected to an air (evacuating or pressuring) pump. The first andsecond needles may be joined in parallel alignment. See, for example,FIG. 24.

The vessel in which a pre-determined quantity of pulsing agent isalready supplied allows diagnostic tests to be performed on individualswithout the necessity for apparatus for measuring out said antigen.Furthermore, when diagnostic test are performed outside laboratoryconditions, problems with contamination and dispensing accuracy can leadto false results in a quantitative assay. A vessel as described hereinovercomes these problems.

Another aspect of the invention is a control vessel, that may be thevessel as described above, except instead of containing a biologicalpulsing agent, it contains a control pulsing agent (control substance).For the purposes of the present description, and as mentioned elsewhere,the vessel as described herein is used to hold pulsing agent and issometimes also called the “reaction vessel”, while the control vessel asdescribed herein is used to hold control pulsing agent. The vessel andcontrol vessel operate independently; they independently receive sampleand stabilizing agent, and interiors are not connected.

However, the vessel and the control vessel may be present together aspart of a kit. Alternatively, the exterior surfaces of the vessel andcontrol vessel may be mechanically connected to form a single entitycomprising both a vessel and control vessel. According to one aspect ofthe invention, the control vessel containing the control pulsing agentis sealed. The control vessel may have a resealing means such as ascrew-cap, push-on cap, a flip-cap for example. The control vessel mayhave a breakable seal such as a peel-back adhesive seal, a snap-offseal. According to one aspect of the invention the wall of the controlvessel may be provided with a resealable area such as a septum, throughwhich substances may be introduced into the control vessel by puncture,using a syringe needle. The septum of the control vessel may beresealable after puncture. According to one aspect of the invention, thebiological sample or other liquids are introduced by means of a transfertubing described above having disposed with a needle either end (e.g. acannular needle). One needle is configured to puncture a septum of atube holding the sample tube or other container, and the other needle isconfigured to puncture the septum of the control vessel; liquidbiological sample is able to be transferred from the sample tube to thecontrol vessel using the needle. The tubing may be flexible or rigid.The shape of the transfer tubing may be U-shaped.

In one aspect of the invention, the control vessel may have a means todraw liquid biological sample from a biological sample tube when influid connection therewith; examples include but are not limited to asyringe-type plunger, or any means to apply negative pressure.

It will be understood that the control vessel receives a portion of thesame biological sample introduced into the reaction vessel. Thebiological sample may be divided into two portions, one for the controland the other for the reactions vessel. Alternatively, one aliquot ofthe biological sample may be introduced into the reaction vessel and asecond equal aliquot may be introduced into the control vessel.

According to another aspect of the invention, the control vessel isprovided with a sealable septum as described above, adapted to receive afirst needle (cannular) through which liquid biological sample can enterthe control vessel and a second needle through which positive ornegative pressure can be applied to the inside of the control vessel.When negative (vacuum) pressure is applied through the second needle,biological sample can be drawn into the control vessel through the firstneedle. Preferably the first needle is part of the transfer tubingdescribed earlier. The second needle may be part of a pressurizingtubing described below, which is connected to an air (evacuating orpressuring) pump. The first and second needles may be joined in parallelalignment. See, for example, FIG. 24.

The control pulsing agent will depend on the pulsing agent, and theparameter under investigation, as is well understood by the skilledartisan. For instance, when the pulsing agent is peptide, a set ofpeptides, or a pool of peptides, the control pulsing agent may comprisesa peptide or peptides of the same length, with the exception that thesequence of the peptide(s) is randomized and/or is not identical to asegment of an existing protein or peptide. This control pulsing agentwould be incubated with the same whole blood as the pulsing agent, withthe same quantity of agent, same volume of blood, for the same period oftime and at the same temperature. In another example, the pulsing agentmay be a protein therapeutic agent, such as IFN-beta dissolved in anexcipient such as water, phosphate-buffered saline, or saline to thedesired concentration. The control pulsing agent may then be theexcipient. In a further example, the pulsing agent may be an antigen,such as Feld1 dissolved in an excipient such as water,phosphate-buffered saline, or saline to the desired concentration; thecontrol pulsing agent may then be excipient of the antigen.Alternatively, the control vessel may devoid of control agent or isempty in which case an induced immune response, or induced geneexpression, for example, is compared to an untreated sample.

Another aspect of the present invention is related to a vessel asdescribed herein, further comprising a container in which a stabilisingagent is present; the stabilising agent is temporarily prevented fromcoming into contact with the pulsing agent or the biological sampleexposed to said agent.

In one aspect of the present invention, the stabilising agent comprisesa nucleic acid stabilising agent and/or cellular RNA degradationinhibiting agent and/or a gene induction inhibiting agent, and/or thestabilising agent is as that found in a PAXgene™ Blood RNA Tube and/orstabilising agent is as that found in a Tempus™ Blood RNA Tube. Agentsand combinations thereof are known in the art, or can be deduced by theskilled artisan.

The PAXgene™ and Tempus™ Blood RNA Tubes are supplied with a solutioncontaining an additive that stabilises cellular RNA and may eliminate exvivo induction of the gene transcription. No detailed information isprovided describing the nature of this additive. The brochure providedwith PAXgene™ tubes refers to U.S. Pat. No. 5,906,744 for this purpose.Nevertheless, the tube described in this patent allows a person skilledin the art to prepare nucleic acids from plasma and not from whole bloodas performed in the present invention. In particular, the device of U.S.Pat. No. 5,906,744 preferably comprises a plastic or glass tube, a meansfor inhibiting blood coagulation and a means for separating plasma fromwhole blood (U.S. Pat. No. 5,906,744 column 2, I.42-43). Therefore,according to the present invention, the content as described in U.S.Pat. No. 5,906,744 does not relate to the real content of the PAXgene™Blood RNA Tube as it relates to a different use.

According to the present invention the solution held in the PAXgene™Blood RNA Tubes may contain a quaternary amine surfactant. Therefore,according to the present invention, a quaternary amine surfactant may beused as a stabilising agent. The use of a quaternary amine surfactant inorder to stabilise nucleic acids in a biological sample has beenpreviously described in U.S. Pat. No. 5,010,183. This patent provides amethod for purifying DNA or RNA from a mixture of biological materials.Said method comprises the step of adding a cationic detergent to amixture containing the RNA or DNA in an amount sufficient to dissolvecells, solubilize any contaminating proteins and lipids in the mixture,and form insoluble hydrophobic complex between the nucleic acid and thedetergent. The complex which comprises the RNA or DNA with the detergentthus becomes separated from the solubilised contaminants. In a morerecent patent, the same inventors stated that the use of the surfactant,as described in U.S. Pat. No. 5,010,183, and other commerciallyavailable surfactants results in inefficient precipitation of RNA andincomplete lysis of blood cells. As there was a need for improvedcationic surfactants for this purpose, the inventors of U.S. Pat. No.5,010,183 searched for a novel method for isolating RNA from abiological sample, including blood, involving the use of an aqueous,cationic surfactant solution comprising a selected quaternary amine(U.S. Pat. No. 5,985,572). New aqueous quaternary amine surfactants,able to stabilize RNA from biological samples, are also described inWO94/18156 and WO02/00599. The synthesis of the different possiblesurfactants, that can be used in any methods of the present invention,can be performed according to the instructions as published in abovecited or related patents. One example of a quaternary amine which can beused in the method of the present invention istetradecyltrimethyl-ammonium oxalate. (U.S. Pat. No. 5,985,572).Alternatively, said cationic detergent may be Catrimox-14™ (U.S. Pat.No. 5,010,183) as shown in the example 1 of the present invention.Further to the stabilization of said biological sample, saidapplications describe the isolation of the nucleic acids usingconventional separation techniques such as column chromatography. Due tothe obliged combination of the PAXgene™ Blood RNA Tube with the PAXgene™Blood RNA kit (which also applies column chromatography) the suppliergives the impression that the compounds present in the PAXgene™ BloodRNA Tube may only be compatible with said chromatographic method.

In one aspect of the present invention, the stabilising agent iscontained in said container until such as time as the biological samplehas mixed with the pulsing agent and/or a user requires introduction ofthe stabilising agent.

The container may be integrated into the vessel, meaning that it thecontainer may be mechanically joined to the vessel so forming aone-piece unit. According to one aspect of the invention, the inside ofsaid container and the inside of said vessel are connected, and aphysical barrier that blocks the connection is present. At anappropriate time, an application of force opens the physical barrier,allowing the stabilising agent to mix with the biological sampleso-pulsed. According to one aspect of the invention a physical barrierreversibly opens and closes in accordance with the physical forceapplied. The force applied may transmit to the physical barrier itself,or via the stabilising agent to the physical barrier. A physical barriermay be any mechanical barrier. Examples of such physical barriersinclude rotary valve, aperture valve, slit valve, diaphragm valve, ballvalve, flap valve. According to another aspect of the invention, thephysical barrier may be irreversibly opened by the application of force.A physical force may be any mechanical force. The force applied maytransmit to the physical barrier itself (see for example, FIG. 7), orvia the stabilising agent to the physical barrier (see for example, FIG.8). Another example of such physical barriers include a plug which isforced out of position (See, for example, FIG. 1), a barrier whichshatters upon the application of force (see, for example, FIG. 7).

According to another aspect of the invention, the inside of saidcontainer and the inside of said vessel are connected, and the flow ofstabilising agent from the container to the vessel is prevented by thesurface tension of the stabilising agent in combination with theaperture size of the connection. According to this aspect of theinvention, at an appropriate time an application of force whichtransmits to the stabilising agent, forces the stabilising agent fromthe container into the vessel. The force may be applied, for example, bysqueezing, continually inverting, and agitating.

Another aspect of the present invention is related to a control vesselas described herein, further comprising a control container in which astabilising agent is present; the stabilising agent is temporarilyprevented from coming into contact with the control pulsing agent or thebiological sample exposed to said control pulsing agent. The vessel withintegrated container described herein, together with the control vesselwith integrated control container may be present together as part of akit. Alternative, the exterior of the vessel with integrated containerdescribed herein, together with the exterior of the control vessel withintegrated control container may be joined or connected using a bridgingmember to form a single device (FIGS. 19 and 20).

The control container may be integrated into the control vessel, meaningthat it the control container may be mechanically joined to the controlvessel so forming a one-piece unit. According to another aspect of theinvention, the inside of said control container and the inside of saidcontrol vessel are connected, and a physical barrier that blocks theconnection is present. At an appropriate time, an application of forceopens the physical barrier, allowing the stabilising agent to mix withthe biological sample so-pulsed. According to one aspect of theinvention a physical barrier reversibly opens and closes in accordancewith the physical force applied. The force applied may transmit to thephysical barrier itself, or via the stabilising agent to the physicalbarrier. Examples of such physical barriers include rotary valve,aperture valve, slit valve, diaphragm valve, ball valve, flap valve.According to another aspect of the invention, the physical barrier maybe irreversibly opened by the application of force. The force appliedmay transmit to the physical barrier itself (see for example, FIG. 17),or via the stabilising agent to the physical barrier (see for example,FIG. 18). Another example of such physical barriers include a plug whichis forced out of position (See, for example, FIG. 12), a barrier whichshatters upon the application of force (see, for example, FIG. 17).

According to another aspect of the invention, the inside of said controlcontainer and the inside of said control vessel are connected, and theflow of stabilising agent from the control container to the controlvessel is prevented by the surface tension of the stabilising agent incombination with the aperture size of the connection. According to thisaspect of the invention, at an appropriate time an application of forcewhich transmits to the stabilising agent, forces the stabilising agentfrom the control container into the control vessel. The force may beapplied, for example, by squeezing, continually inverting, andagitating. The vessel or control vessel as described herein, whichcomprises a container or control container respectively for dispensing astabilising agent, allows an untrained technician to pulse blood using apulsing agent or control, and stabilise the blood so pulsed for analysisby a skilled artisan at a later stage. Thus, where many samples arerequired to be collected, a vessel or control vessel as disclosed hereinallows a cost saving since unskilled operators can be employed to pulseand stabilise the blood. Furthermore, the vessel or control vessel allowreproducibility because known amounts of pulsing agent and stabilisingagent may be pre-supplied in said vessel, so minimising errorsassociated with pipetting. Furthermore, the time between withdrawing abiological sample and exposing said sample to a pulsing antigen orcontrol is greatly reduced since the sample can be drawn directly intosaid tube, or via a syringe for example. Furthermore, the time betweenpulsing the biological sample and stabilising the biological sample canbe accurately set, since the introduction of stabilising agent to thesample is achieved simply by the application of force; hence there areno delays due to pipetting stabilising agent.

Another embodiment of the present invention is a kit suitable forpulsing a biological sample with a pulsing agent, and subsequentlyintroducing a stabilising agent thereto and testing the RNA componentsin the biological sample so pulsed, comprising one or more vessels asdisclosed above and one or more containers in which said stabilisingagent is present. The kit may further comprise a control vessel in whicha biological control pulsing agent is present.

In one embodiment of the kit, the container in which said stabilisingagent is present is not integrated into the vessel in which the pulsingagent in present, and/or the control container in which said stabilisingagent is present is not integrated into the control vessel in which thecontrol substance is present.

The container or control container can thus be separate and may be anycontainer of the art, suitable for holding a stabilising agent in a kit.According to one aspect of the invention, the container or controlcontainer containing the stabilising agent is sealed. The container orcontrol container may have a resealing means such as a screw-cap,push-on cap, a flip-cap for example. The container or control containermay have a breakable seal such as a peel-back adhesive seal, a snap-offseal. According to one aspect of the invention the wall of the containeror control container may be provided with a resealable area such as aseptum; stabilizing agent may drawn from the container or controlcontainer by puncture, using a syringe needle. The septum of thecontainer or control container may be resealable after puncture.According to a particular embodiment, the container or control containeris used in conjunction with a transfer tubing disposed with a needleeither end (e.g. a cannular needle). One needle is configured topuncture a septum of the container or control container and the otherneedle is configured to puncture the septum of the vessel or controlvessel (described below) respectively; stabilising agent is able to betransferred from the container or control container to the vessel orcontrol vessel respectively using the transfer tubing. The tubing may beflexible or rigid. The shape of the transfer tubing may be U-shaped. Thecontainer or control container might comprise one or more fittingssuitable for attachment of said vessel fitted reciprocal coupling meansand transfer of stabilising agent to said vessel or control vessel. Forexample, the container or control container might be fitted with a Luerfitting that can receive a reciprocal Luer fitting attached to saidvessel as described above (see, for example, FIGS. 9, 10 and 11), orattached to the control vessel. In another example, the container orcontrol container might be fitted with a non-Luer fitting which can matewith a vessel or control vessel having a reciprocating non-Luer designof coupling. The stabilising agent might be transferred to the vessel orcontrol vessel by opening the resealing means of the vessel; thestabilising agent may exit the container or control container via any ofthe aforementioned fittings or openings. The container or controlcontainer may optionally have a means to allow air to enter whilestabilising agent exits. In one aspect of the invention, the containeror control container may have a means to force stabilising agent fromsaid container or control container; examples include but are notlimited to a syringe-type plunger, squeezable walls of the container orcontrol container or any means to apply positive pressure. The containeror control container optionally has a measuring means to determine thevolume of stabilising agent being dispensed, for example, a scale. Inone aspect of the invention, the container or control container holds avolume of stabilising agent sufficient for single use. In another aspectof the invention, the container or control container holds a volume ofstabilising agent sufficient for multiple pulsing experiments.

According to another aspect of the invention, the container or controlcontainer is provided with a sealable septum as described above, adaptedto receive a first needle (cannular) through which stabilizing agent canexit the vessel and a second needle through which positive or negativepressure can be applied to the inside of the vessel. When positivepressure is applied through the second needle, stabilising agent can bepropelled from the container or control container through the firstneedle. Preferably the first needle is part of the transfer tubingdescribed earlier. The second needle may be part of a pressurizingtubing described below, which is connected to air (evacuating orpressuring) pump. The first and second needles may be joined in parallelalignment.

In another embodiment of the kit, the container in which saidstabilising agent is present is connected to the vessel in which thepulsing agent is present; embodiments of the vessel are described above.In another embodiment of the kit, the control container in which saidstabilising agent is present is connected to the control vessel in whichthe control substance is present; embodiments of the control vessel aredescribed above.

Optionally, a kit of the present invention may comprise an instructionmanual comprising a description of a method for pulsing a biologicalsample.

Another aspect of the present invention is related to a vessel, and akit comprising said vessel as disclosed herein wherein said pulsingagent comprises an antigen. According to one aspect of the inventionsaid antigen is bacterial LPS. According to another aspect of theinvention said antigen is an immune response recall antigen. Accordingto another aspect of the invention said antigen is tetanous toxoid.According to another aspect of the invention said antigen is a cancerimmunotherapy antigen. According to another aspect of the invention saidantigen is MAGE-3. According to another aspect of the invention saidantigen is a cat allergen. According to another aspect of the inventionsaid antigen is Feld1. According to another aspect of the invention saidantigen is antigen presenting cells from an organ donor. According toanother aspect of the invention said antigen is an autoantigen.According to another aspect of the invention said antigen is GAD65.

The control pulsing agent will depend on the pulsing agent, and theparameter under investigation, as is well understood by the skilledartisan. For instance, when the pulsing agent is peptide, a set ofpeptides, or a pool of peptides, the control pulsing agent may comprisesa peptide or peptides of the same length, with the exception that thesequence of the peptide(s) is randomized and/or is not identical to asegment of an existing protein or peptide. This control pulsing agentwould be incubated with the same whole blood as the pulsing agent, withthe same quantity of agent, same volume of blood, for the same period oftime and at the same temperature. In another example, the pulsing agentmay be a protein therapeutic agent, such as IFN-beta dissolved in anexcipient such as water, phosphate-buffered saline, or saline to thedesired concentration. The control pulsing agent may then be theexcipient. In a further example, the pulsing agent may be an antigen,such as Feld1 dissolved in an excipient such as water,phosphate-buffered saline, or saline to the desired concentration; thecontrol pulsing agent may then be excipient of the antigen.Alternatively, the control vessel may devoid of control agent or isempty in which case an induced immune response, or induced geneexpression, for example, is compared to an untreated sample.

Another aspect of the present invention is related to a method ofpulsing a biological sample with an antigen, and subsequentlystabilising the nucleic acid therein and testing the RNA components inthe stabilising biological sample so pulsed. The method comprises theuse of a vessel, control vessel and/or kit as disclosed herein tooptionally collect, pulse and stabilise the sample.

One embodiment of the invention is a device for accepting a liquidbiological sample, which facilitates exposure of said sample separatelyto a first substance and a control substance, and subsequently to anucleic acid stabilizing agent, the device comprising:

-   -   a first compartment where a first substance is present,    -   a second compartment in which a control substance is present,    -   a third compartment where a stabilizing agent is present, and    -   a support for a biological sample tube.

An example of a device is shown in FIGS. 21 a and 21 b. According to oneaspect of the invention, at least one, (e.g. 1, 2, 3, all), preferablyall the compartments are sealed. One or more (e.g. 1, 2, 3, all)compartments may have a resealing means such as a screw-cap, push-oncap, a flip-cap for example. One or more (e.g. 1, 2, 3, all)compartments may have a breakable seal such as a peel-back adhesiveseal, a snap-off seal. According to one aspect of the invention one ormore (e.g. 1, 2, 3, all) compartments is provided with a resealable areasuch as a septum, through which substances may be introduced into thecompartment by puncture, using a syringe needle. The septum of thecompartment may be resealable after puncture.

According to a particular embodiment, one or more (e.g. 1, 2, 3, all)compartments and the biological sample tube is used in conjunction witha transfer tubing—a hollow tubing disposed with a hollow needle eitherend (e.g. a cannular of hypodermic needle) as shown, for instance, inFIG. 22. One needle is configured to puncture a septum of onecompartment (e.g. the first compartment) and the other needle isconfigured to puncture the septum of the other compartment (e.g. thethird compartment). Alternatively, one needle is configured to puncturea septum of one compartment (e.g. the first compartment) and the otherneedle is configured to draw fluid from the biological sample tube (heldby the support); in this way, two compartments or one compartment andthe biological sample tube can be temporarily fluidicly connected viathe tubing. The fluid connection allows stabilising agent, for example,to be transferred from the third compartment to the first and/or secondcompartment using the transfer tubing. The tubing may be flexible orrigid. The transfer tubing may be U-shaped.

The biological sample tube according to the invention can be anysuitable for storage of a biological sample. According to one aspect ofthe invention, the biological sample tube is sealed. According to oneaspect of the invention, the biological sample tube has a resealingmeans such as a screw-cap, push-on cap, a flip-cap. According to oneaspect of the invention, a biological sample may be introduced into thetube by puncture, using a syringe needle into the wall of the tube. Thewall of the tube may be resealable after puncture, or the wall of thetube may not be resealable after puncture, or the wall of the sampletube may be provided with a resealable area such as a septum. Accordingto one aspect of the invention, the biological sample may be drawn fromthe tube introduced by means of a transfer tubing comprising a hollowtube disposed with a needle either end (e.g. a cannular needle) asdescribed elsewhere herein. One needle is configured to puncture aseptum of a sample tube holding the sample or other liquids, and theother needle is configured to puncture the septum of a compartment;liquid biological sample is able to be transferred from the sample tubeto one of the compartments using the tubing. The tubing may be flexibleor rigid. The shape of the transfer tubing may be U-shaped. According toone aspect of the invention, the biological sample may be introducedinto the sample tube by means of one or more fittings attached to sampletube adapted to a syringe or other container fitted with a couplingmeans. For example, the tube might be fitted with a Luer fitting thatcan receive a needleless syringe. In another example, the tube might befitted with a non-Luer fitting which can mate with a container having areciprocating non-Luer design of coupling. According to one aspect ofthe invention, the biological sample may be introduced into the tube bymeans of a cannular or hypodermic needle fitted to said tube, suitablefor directly withdrawing biological samples from an individual.According to one aspect of the invention, the biological sample may beintroduced into the sample tube by opening the resealing means.

In one aspect of the invention, the first compartment may have a meansto draw liquid biological sample from a biological sample tube into thefirst compartment when in fluid connection therewith. The secondcompartment may similarly have a means to draw liquid biological samplefrom a biological sample tube into the second compartment when in fluidconnection therewith using the transfer tubing for instance. Examples ofsuch means include but are not limited to the use of a syringe-typeplunger, or any means to apply negative pressure to the receivingcompartment, or positive pressure to the supplying compartment.According to a preferred embodiment of the invention, pressure insidethe compartment is controlled using an insertable pressurising tubingdescribed below. The first and/or second compartments may partially heldunder vacuum.

In one aspect of the invention, the first compartment may have a meansto draw stabilising agent from the third compartment into the firstcompartment when in fluid connection therewith. The second compartmentmay similarly have a means to draw stabilising agent from the thirdcompartment into the second compartment when in fluid connectiontherewith using the transfer tubing for instance. Examples of such meansinclude but are not limited to the use of a syringe-type plunger, or anymeans to apply negative pressure to the receiving compartment, orpositive pressure to the supplying compartment. According to a preferredembodiment of the invention, pressure inside the compartment iscontrolled using an insertable pressurising tubing described below. Thefirst and/or second compartments may partially held under vacuum.

In another aspect of the invention, the third compartment may have ameans to propel stabilising agent from the third compartment into thefirst and/or into the second compartment when in fluid connectiontherewith using the transfer tubing for instance. Examples of such meansinclude but are not limited to a syringe-type plunger, or any means toapply positive pressure to the third compartment. According to apreferred embodiment of the invention, pressure inside the compartmentis controlled using an insertable pressurizing tubing described below.The third compartment may be held under positive pressure.

According to one aspect of the invention, the first compartment issterile. According to another aspect of the invention, the secondcompartment is sterile. According to another aspect of the invention,the third compartment is sterile. According to another aspect of theinvention, all the compartments are sterile.

According to one embodiment of the invention, the each compartment isprovided with a sealable septum as described above, adapted to receive afirst needle through which liquid can pass and a second needle throughwhich positive or negative pressure can be applied to the inside of thecompartment. When positive pressure is applied through the secondneedle, liquid can be propelled from the compartment through the firstneedle. Alternatively, when negative pressure is applied through thesecond needle, liquid can be draw into the compartment through the firstneedle. Preferably the first needle is part of the transfer tubingdescribed earlier. The second needle is part of the pressurising tubing.The first and second needles may be joined in parallel alignment. See,for example, FIG. 24.

Where necessary, one or more (e.g. 1, 2, 3, all) compartments mayoptionally be disposed with a vent that allows pressure in thecompartment to be equalised with ambient pressure. The vent may comprisea one way valve (e.g. rotary valve, aperture valve, slit valve,diaphragm valve, ball valve, flap valve) configured to provide a passageof gas in only one direction, for example, it may allow the release ofexpelled gas.

According to a further aspect of the invention, the device is furtherprovided with a support for the biological sample tube, also knownherein as a sample tube support. The sample tube support may be anysuitable structure, mechanical or otherwise, that holds the sample tubein an upright position. In a preferred embodiment, the sample tubesupport is a walled aperture having a shape reciprocal to at least thebase region of the sample tube.

According to a further aspect of the invention, the device is furtherprovided with a support for a transfer tubing, also known herein as atubing support. The tubing support may be any suitable structure,mechanical or otherwise, adapted for demountable attachment of thetransfer tubing to the device. The tubing support allows storage of thetransfer tubing during transport and non-use. It also provides a cleanenvironment for the needle ends of the transfer tube. In a preferredembodiment, the transfer tubing support comprises two slots each havinga shape reciprocal to each end of the transfer tubing.

The device may take any form which provides the requirements for thecompartments and supporting elements. It will be understood that itshould preferably be optimized for size, weight, supporting ability,durability and recyclability. In a preferred embodiment of theinvention, the compartments are mechanically connected to each other.Preferably, the sample tube support is also mechanically connected tothe compartments. Preferably, the tubing support is also mechanicallyconnected to the compartments. Being so connected, the device is asingle entity that has the convenience of being an autonomous unit.Mechanical connection may be achieved using any suitable arrangement asknown in the art, for example, employing compartments formed from asingle block of biologically inert material such as polycarbonate,polypropylene, cyclic olefin copolymer (COC) or glass mechanicallyconnected to a support of the same or different material.

A preferred embodiment of the invention is shown in three dimensionalview in FIG. 21 a and in cross-section in FIG. 21 b, which depict adevice 30 formed from a solid block 44 of a material such as cyclicolefin copolymer (COC), provided with a first compartment 32 in which afirst substance 2 is present, a second compartment 34 in which a controlsubstance 35 is present, and a third compartment 36 in which astabilizing agent 5 is present. The compartments 32, 34, 36 arecylindrical openings provided in the solid block, 44 each sealed with alid 46, 48, 50 which may also be made at least partly from COC.Alternatively, the compartments 32, 34, 36 may each be formed from acylindrical vial made from a suitable material (e.g. glass,polypropylene, polycarbonate) fixed in the cylindrical openings providedin the solid block, 44, each vial each sealed with a lid 46, 48, 50.Each lid 46, 48, 50, is disposed with a resealable septum 38, 40, 42,providing access to the compartment interior using a needle. A sampletube 52 support is provided on the device. Said sample tube support 52is a cylindrical opening configured to support the base region of asample tube. The device is further disposed with a tubing support 54, 56comprising two slots each having a shape reciprocal to each end of thetransfer tubing. Using a U-shaped transfer tubing, the contents of thesample tube can be transferred to each of first (pulsing agent)compartment 32 and second (control) compartment 34, said compartmentsbeing under negative pressure. The same needle may be used to transferthe stabilizing agent from the third compartment 36 into each of thefirst 32 and second 34 compartments.

As described elsewhere, a transfer tubing comprising a hollow tubedisposed with a needle at either end (e.g. a cannular needle) is used totransfer liquid from one compartment or vessel to another, the liquideither being propelled by the application of positive pressure to theproviding compartment (or container or control container), or beingdrawn by application of negative pressure in the receiving compartment(or vessel or control vessel,).

The transfer tubing needles are suited to piercing a resealable (e.g.rubber or silicone) septum so that fluid or air may only pass into thecompartment (vessel or control vessel) through the needle. The pointedend of the needle is preferably beveled with both sides sharp, and maybe non-coring. The needle may be 22 G, 23 G, 24, G or 25 G, or a valuein the range between any two of the aforementioned values, preferably 23G. Each needle may be protected with a detachable water-impermeablecover to prevent the ingress of micro-organisms prior to use. Eachneedle cover may reside in and be attached to the tubing support suchthat when the U-shaped needle is lifted from the device, the coverdetaches from each needle, leaving the cover still within the tubingsupport.

The length (L) of the transfer tubing needles in the transfertubing—which refers to the length of the straight part of the needle—maybe the same or different. Their lengths will depend on the depth of thecompartments and on the volume of liquid that is to be transferred. As ageneral guidance, the length (L1) of one transfer tubing needle may be25, 26, 28, 30, 32, 33, 34, 35, 36, 38, 40, 42, 44, 46, 48, 50, 55 mm,or a value in the range between any two of the aforementioned values,preferably between 25 and 35 mm, more preferably between 30 and 35 mm.The length (L2) of the other transfer tubing needle may be 25, 26, 28,30, 32, 33, 34, 35, 36, 38, 40, 42, 44, 46, 48, 50, 55 mm, or a value inthe range between any two of the aforementioned values, preferablybetween 40 and 50 mm, more preferably between 42 and 46 mm. The heightof the tubing (L3) will depend on the clearance space available. As ageneral guidance, the length (L3) of one transfer tubing may be 5, 7, 8,9, 10, 11, 12, 13, 14, 15, 16 or 17 mm, or a value in the range betweenany two of the aforementioned values, preferably between 10 and 15 mm,more preferably between 12 and 14 mm. The hollow tubing may be rigid orflexible. Preferably it is rigid. According to a preferred aspect of theinvention, the transfer tubing is formed from a long needle, bent in a Uform, whereby a second needle is attached to the non-pointed end so asto form the transfer tubing of the invention, having a rigid hollowtube. The distance (D) between both needles where rigid tubing isemployed will depend on the spacing between the compartments. Accordingto a preferred aspect of the invention, the distance (D) between bothneedles is essentially the same as the distance between two adjacentcompartments; this is generally achieved when the compartments andsample support are arranged in a square 2×2 array. As a generalguidance, the distance between the needles may be 20, 22, 24, 26, 28,30, or 32 mm, or a value in the range between any two of theaforementioned values, preferably between 24 and 28 mm, more preferablybetween 26 mm.

The transfer tubing may be adapted for attachment to a robotic system(e.g. robotic arm or platform) to facilitate automated sampleprocessing. Where the rigid hollow tubing is employed, the roboticsystem is attached to the transfer tubing via the rigid hollow tubing.The use of a robotic system permits the automated assays to be performedusing several devices of the invention, also in a sterile environment,potentially devoid of other stimulating agents. Moreover, the device canfacilitate transfer of liquid reagents by utility of a single transfertubing.

The pressure to a compartment, vessel, or container may be provided by ahollow pressurizing tubing (FIG. 23) comprising a flexible tubing havinga needle disposed at one end for insertion into the septum of acompartment, vessel, or container, the other end adapted for attachmentto an air pump that can provide the requisite controlled pressure to thecompartment, vessel or container. This pressurizing tubing needle may bemechanically attached to one of the needles of the transfer tubing by ajoint such as a welded joint, adhesive joint, or clamp (see FIG. 24).The pressurizing tubing needle is suited to piercing a resealable (e.g.rubber or silicone) septum so that air or gas may only enter or leavethe compartment, vessel or container through the needle. The pointed endof the needle is preferably beveled with both sides sharp, and may benon-coring. The needle may be 22 G, 23 G, 24, G or 25 G, or a value inthe range between any two of the aforementioned values, preferably 23 G.

It is within the scope of the invention that the device is disposed withadditional sealed compartments. The compartments may be arranged along astraight line or in an array (e.g. 2×2, 2×4 etc). Preferably, thecompartments and sample support are arranged in a 2×2 square array, thedistance between adjacent compartments and sample support being equal.

In one embodiment of the present invention, a method of pulsing abiological sample comprises the steps of:

i) introducing a biological sample into said vessel,

ii) optionally agitating said vessel,

iii) introducing stabilising agent into said vessel after apre-determined period of time, and

iv) testing the levels of nucleic acid therein.

Another embodiment of the present invention is method of testing theimmune response of an individual towards an antigen against which theindividual has been pre-immunised comprising the use of a vessel asdisclosed herein wherein the pulsing agent is the antigen underinvestigation and the steps of:

a) introducing a sample of blood taken from said individual into thevessel,

b) optionally agitating said vessel,

c) after a pre-determined period of time, introducing said nucleic acidstabilising agent into said vessel

d) testing the levels of cytokine mRNA.

According to one aspect of the invention the cytokine is one or more ofIL-2, IL-4, IL-13, IFN-gamma.

Another embodiment of the present invention is method of testing anindividual for hyperallergenicity towards an antigen comprising the useof a vessel as disclosed herein wherein the pulsing agent is the antigenunder investigation and the steps of:

e) introducing a sample of blood taken from said individual into thevessel,

f) optionally agitating said vessel,

g) after a pre-determined period of time, introducing a nucleic acidstabilising agent into said vessel

h) testing the levels of IL-4 mRNA.

Another embodiment of the present invention is method of testing anindividual for rejection of an organ transplant comprising the use of avessel as disclosed herein wherein the pulsing agent is ahistocompatibility antigen of the donor and the steps of:

i) introducing a sample of blood taken from said individual into thevessel,

j) optionally agitating said vessel,

k) after a pre-determined period of time, introducing a nucleic acidstabilising agent into said vessel

l) testing the levels of IL-2 mRNA.

The methods describe above may be adapted for use with the device of theinvention, for instance, one embodiment of the present invention, is amethod of pulsing a liquid biological sample present in a biologicalsample tube using a device of the invention comprising the steps of:

i) transferring liquid biological sample from the biological sample tubeto the second compartment in which control agent is present, using the Ushaped needle,

ii) transferring liquid biological sample from the biological sampletube to the first compartment in which pulsing agent is present, usingthe U shaped needle,

iii) transferring stabilizing agent from the third compartment to thebiological sample tube, using the U shaped needle,

iv) transferring stabilizing agent from the third compartment to thesecond compartment in which control agent and liquid biological sampleare present, using the U shaped needle,

v) transferring stabilizing agent from the third compartment to thefirst compartment in which pulsing agent and liquid biological sampleare present, using the U shaped needle, thereby pulsing the liquidbiological sample with a pulsing and control agent, and stabilizing thesample so-pulsed.

The other methods described above, namely of testing the immune responseof an individual towards an antigen against which the individual hasbeen pre-immunised, of testing an individual for hyperallergenicitytowards an antigen, of testing an individual for rejection of an organtransplant may be similarly adapted for use with the device of theinvention.

Another aspect of the invention relates to a method of pulsing abiological sample with an antigen, and subsequently stabilising thenucleic acid therein and testing the RNA components in the stabilisingbiological sample so pulsed, comprising the steps of:

A) Pulsing said biological sample with a pulsing agent and adding acompound inhibiting RNA degradation and/or gene induction thereto, usinga kit, device and/or method as disclosed above,

B) forming a precipitate comprising nucleic acids,

C) separating said precipitate of step (B) from the supernatant,

D) dissolving said precipitate of step (C) using a buffer, forming asuspension,

E) isolating nucleic acids from said suspension of step (D) using anautomated device,

F) dispensing/distributing a reagent mix for RT-PCR using an automateddevice,

G) dispensing/distributing the nucleic acids isolated in step (E) withinthe dispensed reagent mix of step (F) using an automated device, and,

H) determining the in vivo levels of transcripts using the nucleicacid/RT-PCR reagent mix of step (G) in an automated setup.

The above methods may optionally employ a control described herein towhich the biological sample is exposed. The control contains containingno reagent or contains a control pulsing agent. The method is performedessentially at the same time as exposure to the pulsing agent, and in anidentical way to the method employing the vessel. The results of thecontrol vessel may be used to normalize the results obtained from thevessel.

Inhibition of RNA degradation and/or gene induction at the moment of thebiological sampling is crucial in order to retrieve a pool of RNAs whichcan be used to determine the in vivo transcript levels. Cellular RNA canbe purified using the PAXgene™ Blood RNA System in its complete form,however, the present invention proves that real in vivo levels can notbe measured using this system ‘as such’ (see example 2).

The present invention shows that the in vivo levels of nucleic acidtranscripts can only be measured/determined/quantified when startingfrom a pool of RNA prepared from a stabilised biological sample, using acompound inhibiting extra- and/or intracellular RNA degradation and/orgene induction; whereby the isolation of the nucleic acids is performedusing an automated device, whereby the reagent mix and the isolatednucleic acids, used for the RT-PCR reaction, are dispensed using anautomated device, and whereby the determination of the transcript levelsis performed in an automated setup. According to the present invention,only this approach allows the quantification of in vivo RNA in areproducible manner. The number of steps performed in said method isreduced to a minimum in order to avoid errors. An ‘error’ may be apipetting-, a handling-, a procedural- and/or a calculation error or anyerror which can be made by a person skilled in the art. In this respect,the present invention suggests to perform the RT and the PCR reaction inone step. The method of the present invention will even be more accuratewhen combining more intermediate steps. For example, in the method ofthe present invention steps (A) and (B) can be combined.

In another aspect of the present invention, the dispension of thenucleic acids (step (G)) may be performed after, before orsimultaneously with the dispension of the reagent mix needed for RT-PCR(step (F)).

According to the method of present invention, OD measurements do notneed to be performed, eliminating the errors made in the calculation ofthe nucleic acid concentration. In contrast, using the complete PAXgene™Blood RNA kit OD measurements need to be made. This illustrates againthat the method according to present invention is a more reliable andaccurate method compared to the latter system. This better accuracy ofthe present invention is illustrated by the reproducibility studiespresented in Table 1.

In another aspect of the present invention, when dissolving the formedprecipitate in step (D) of the method according to the presentinvention, the obtained suspension can be used in combination with anRNA extraction method and an analyzing method which are fully automated.It is only this combination which allows the accurate optimisation andreproducibility of the performed method and which allows the accurateand reproducible determination of RNA levels after pulsing. As thebrochure of the PAXgene™ Blood RNA System describes that thecorresponding tubes can not be used in combination with other isolationmethods, and no detailed information is available describing thedifferent compositions of the kit, it is not obvious for a personskilled in the art to use parts of this PAXgene™ Blood RNA System anddevelop a new method therefrom.

There exist only few commercial systems which allow the isolation of RNAfully automatically. Examples of such automated nucleic acid extractorsare: the MagNA Pure LC Instrument (Roche Diagnostics), The AutoGenprep960 (Autogen), the ABI Prism™ 6700 Automated Nucleic Acid Workstation(Applied Biosystems), WAVE® Nucleic Acid Analysis System with theoptional WAVE® Fragment Collector FCW 200 (Transgenomic) and theBioRobot 8000 (Qiagen).

The present invention points towards the fact that for all these systemsit is essential to start with material which is as fresh as possible orwhich is stabilised in order to allow the determination transcript levelafter pulsing, wherein RNA degradation is minimised. The problem for allthese systems is that the biological sample is collected and brought tothe laboratory in tubes that contain no or only a conventional additive,so that mRNA can still be rapidly degraded. Consequently, mRNAquantification using these methods will undoubtedly lead to thequantification of the transcripts present in the tube, but thisquantification does not represent the transcript levels present in thecells/biological agent at the moment of sampling. Experimental evidenceof this is provided in FIG. 32.2 of example 1 of the present invention.

The term ‘quantification’ is meant accurate and reproducibledetermination of RNA copy numbers; but it is trivial for a personskilled in the art that also qualitative or semi-quantitative studiescan be performed using RNA isolated via a method as described by thepresent invention.

The definition ‘transcript’ is not limited to messenger RNA (mRNA) butalso relates to other types of RNA molecules known to exist by a personskilled in the art. According to the method of the present inventionmRNA as well as total RNA can be extracted. This allows to get a correctestimation of the in vivo nuclear RNA, providing a powerful tool toevaluate gene transcription.

The term ‘nucleic acid’ refers to a single stranded or double strandednucleic acid sequence, said nucleic acid may consist ofdeoxyribonucleotides (DNA) or ribonucleotides (RNA), RNA/DNA hybrids ormay be amplified cDNA or amplified genomic DNA, or a combinationthereof. A nucleic acid sequence according to the invention may alsocomprise any modified nucleotide known in the art.

According to the present invention, the nucleic acid may be presentextra- or intracellularly in the biological sample.

The ‘separation’ of the precipitate from the supernatant in step (C) ofpresent method can be performed via centrifugation, filtration,absorption or other means known by a person skilled in the art. Saidprecipitate may include cells, cell/debris, nucleic acids or acombination thereof. The basis of the concept is to stop thenucleic-acid-containing-agent (or biological agent) from having contactwith external sources/pulses/signals. This can be performed by fixing,lysing and/or disintegrating the nucleic-acid-containing-agent, or byany other means known by a person skilled in the art.

The buffer used in step (D) of the method of present invention may be abuffer to dissolve the precipitate obtained in step (C) of said method.This buffer may have additional effects such as lysis or further lysisof the nucleic-acid-containing-agent.

The ‘automated device’ used may be an automated pipetting device oranother automated device known by a person skilled in the art suitablefor carrying out the indicated actions.

With a ‘reagent mix for RT-PCR’ is meant all reagents needed for asimultaneous RT and PCR reaction (with the exception of theoligonucleotides when explicitly mentioned). According to the presentinvention, ‘oligonucleotides’ may comprise short stretches of nucleicacids as found in for example primers or probes. According to thepresent invention, this method can be used in combination withmicro-arrays or RNase protection assays.

As pointed out before, storage of biological samples such as blood leadsto incorrect determination of mRNA levels. Indeed, in practice, theanalysis of fresh sample is not feasible as the place of sampling andthe place of RNA analysis is located differently. The method accordingto the present invention allows the transport biological samples from aremote site to a suitable laboratory without any effect on their in vivotranscript content. Transport of the biological sample can be performedafter step (A) or step (B) in the method of the present invention.

Usually, when using blood samples, red blood cells are preferentiallyeliminated before the nucleic acids are isolated. Red blood cells arerich in haemoglobin and their presence results in the production ofhighly viscous lysates. Therefore, removal of these allows to isolatenucleic acids in a more improved fashion. However, in the method of thepresent invention, this step is eliminated as an insoluble precipitateis immediately formed comprising the nucleic acids, separating thesefrom all other components of the biological sample. This illustratesthat, in addition to other advantages, the method of the presentinvention is a superior method in comparison with most prior artmethods.

According to the present invention, said buffer used in step (D) of amethod of the present invention may be aguanidine-thiocyanate-containing buffer.

In the examples of the present invention the precipitate formed in thePAXgene™ Blood RNA Tubes is dissolved in the lysis buffer as provided bythe MagNA Pure LC mRNA Isolation Kit I (Roche Diagnostics, MolecularBiochemicals). Therefore, it is suggested in the present invention thatone of the possible buffers which may be used in the method of thepresent invention is a guanidine-thiocyanate-containing lysis buffer asprovided by MagNA Pure LC mRNA Isolation Kit I (Roche Diagnostics,Molecular Biochemicals).

The MagNA Pure LC mRNA Isolation Kit I (Roche Diagnostics, MolecularBiochemicals) is especially designed for use on the MagNA Pure LCInstrument, to guarantee the isolation of high quality and undegradedRNA from whole blood, white blood cells, and peripheral bloodlymphocytes. According to its product description, obtained RNA issuitable for highly sensitive and quantitative LightCycler RT-PCRreactions, as well as for standard block cycler RT-PCR reactions,Northern blotting and other standard RNA applications. Nevertheless, thepresent invention proves that the use of this method ‘as such’ could notresult in the determination of correct transcript levels. The presentinvention shows that there is a need to stabilize the RNA prior to theRNA isolation (see example 1). The present invention describes theunique combination of the use of RNA stabilizing compounds and anautomated isolation/analysis procedure.

According to the present invention, once the precipitate of step (D) isdissolved in a lysis buffer such as the one provided by MagNA Pure LCmRNA Isolation Kit I, the method of the present invention may follow theprocedure as described for the MagNA Pure LC mRNA Isolation Kit I. Afterthe samples are lysed through the presence of a chaotropic salt in thelysis buffer, streptavidin-coated magnetic particles are added togetherwith biotin-labeled oligo-dT, and the mRNA binds to the surface of theparticles. This is followed by a DNase digestion step. mRNA is thenseparated from unbound substances using a magnet and several washingsteps. Finally, the purified mRNAs are eluted. This isolation kit allowsthe automated isolation of pure mRNA as a “walk away” system. It allowsto isolate mRNA of high quality and integrity suitable for all majordownstream applications regarding gene expression analysis. Differentprotocols are offered depending on the sample material used. The samplesmay be set directly on the MagNA pure LC Instrument stage. When usingwhole blood, cells present in the samples are preferentially lysedmanually. mRNA isolation may then be postponed or directly furtherprocessed on the instrument.

The present invention proves in the present examples that the use of theMagNA Pure LC Instrument (Roche Diagnostics, Molecular Biochemicals) asautomated device in step (E), step (F) and/or step (G) of the methodaccording to the present invention leads to the production of a pool ofRNA which can be used to determine exact levels of transcripts afterexposure to a pulsing agent. RNA-capturing beads such as magnetic beads,coated with oligo-dT via a streptavidin-biotin system or an equivalentsystem, may be applied in the method of the present invention in orderto separate mRNA from the cellular debris.

Alternatively, according to the present invention other automateddevices may be used such as the ABI Prism™ 6700 Automated Nucleic AcidWorkstation (Applied Biosystems) or any other automated device that canbe used for this purpose.

In the brochure of the MagNA pure LC mRNA Isolation Kit I (Cat No 3 004015) no compositions of the buffers used in this kit are mentioned indetail. Therefore it is not obvious for a person skilled in the art toassume that the buffer as provided by this kit would allow to dissolvethe pellet obtained by the method of the PAXgene™ Blood RNA Tubes. Inaddition, a person skilled in the art would not combine both methodsbased on the information provided by the PAXgene™ Blood RNA Tubesbrochure stating that these tubes can only be combined with thecorresponding PAXgene™ Blood RNA Kit (page 3, see limitations of thesystem; page 6, see ordering information).

As pointed out above, when using blood samples, red blood cells arepreferentially lysed after step (A) in the method of the presentinvention. In the design of the MagNA Pure LC mRNA Isolation Kit I(Roche Diagnostics, Molecular Biochemicals) there is a possibility tolyse and eliminate red blood cells, before mRNA isolation from whiteblood cells. Nevertheless, because of this step, samples cannot betreated fast enough to avoid mRNA degradation. The present inventorsdecided to use the stabilising agent contained in the PAXgene™ Blood RNATube in conjunction with the MagNA Pure mRNA Isolation Kit on the MagNAPure Instrument. Using the PAXgene™ Blood RNA Tubes provides aprecipitate of nucleic acids that is not supposed to be soluble in thelysis buffer of the MagNA Pure mRNA Isolation Kit. Despite of this, theinventors found that it is actually possible. Following thisobservation, the inventors combined the use of the stabilising agent inthe PAXgene™ Blood RNA Tubes with the use of an automated RNA isolationsystem. The inventors found surprisingly that this combination ispossible and that this combination provides a powerful technique for theaccurate mRNA quantification from biological samples.

The RNA isolated using the method according to the present invention isready for use in a wide range of downstream applications, including forinstance nucleic acid amplification technologies, such as RT-PCR andNASBA®, Expression-array and expression-chip analysis, QuantitativeRT-PCR, including TaqMan® technology, cDNA synthesis, RNase and S1nuclease protection, Northern, dot, and slot blot analysis and primerextension.

The present inventors showed in the example 1 and example 2 of thepresent invention that the use of a compound inhibiting RNA degradationand/or gene induction in conjunction with an automated RNA isolation andan automated analysis method such as real time PCR allows thedetermination of in vivo levels of transcripts. Nevertheless, accordingto present invention analysis methods other than real-time PCR may beapplied as long as they are provided in an automated setup.

A main advantage of the method according to the present invention, isthe fact that by using this method small sample volumes can be analyzed.This is of prime importance when only small volumes are available, forexample when analyzing neonatal blood samples or in cases of high bloodloss. According to the present invention RNA quantification may beperformed using a biological sample as small as 100 μl. The analysis ofRNA from a sample as small as 100 μl is not possible with the Qiagen kit(PAXgen™ Blood RNA System) which requires a larger volume of blood (2.5ml following the kit handbook).

As mentioned above, one aspect of the present invention is a kitsuitable for pulsing a biological sample with a pulsing agent, andsubsequently stabilising the nucleic acid from the biological sample sopulsed. In another aspect of the invention, the said kit comprisesadditional components for isolating quantifiable RNA from thestabilised, pulsed biological samples. According to an aspect of theinvention, the kit may comprise additional components such as:

-   -   reagents for automated RNA isolation,    -   a reagent mix for a simultaneous RT and real-time PCR reaction        or separate compounds thereof, allowing the automated dispension        of said mix,    -   optionally, specific oligonucleotides to perform said RT-PCT        reactions, and,    -   optionally, an instruction manual describing a method for an        automated RNA isolation, a method for the automated dispension        of a reagent mix and the dispension of the isolated nucleic        acids for RT—real time PCR, and a method for automated RNA        analysis.

In the present examples the present inventors apply the “LightcyclermRNA hybridisation probes kit” from Roche Diagnostics, MolecularBiochemicals (cat #3 018 954) to perform the RT-PCR reactions in onestep. All reagents needed are included in this kit, except theoligonucleotides (synthesized by Biosource). Nevertheless, real time PCRas described in the present invention can also be performed on otherinstruments such as the Applied Biosystems instruments. The kit mayadditionally comprise a buffer such as aguanidine-thiocyanate-containing buffer which can be used in step (b) ofthe method according to the present invention.

The method according to the present invention can also be used for thequantification/detection of DNA (double or single stranded) inbiological samples. Therefore, the present invention also relates to amethod for the quantification of DNA from a biological sample wherein amethod is used as performed for the quantification of RNA according tothe present invention, wherein the RT reaction is skipped and whereinthe compound of step (a) also protects the DNA from being degraded. Asthese nucleic acids are more stable than RNA, its stabilization is lessimportant than for RNA.

In addition, the present invention relates to a kit for isolatingquantifiable DNA from a biological sample according to the presentinvention, wherein a reagent mix/compounds for performing said RTreaction is absent. Situations where exact DNA levels need to bedetermined in biological samples may be to determine the ‘presence’ ofinfection(s)/contamination(s) in biological samples by unexpected genes,pathogens or parasites; and/or to determine the ‘level’ of saidinfection/contamination. For example the method may be used to determinethe percentage of transgenic material in a cereal batch.

The present invention also relates to the use a device, kit and method,according to the present invention, for the monitoring/detection ofchanges of in vivo nucleic acids of a biological marker in a biologicalsample after pulsing with an agent, in order to diagnose a certaindisease.

The present invention also relates to the use a device, kit and methodaccording present invention, for the monitoring/detection of changes ofin vivo nucleic acids of a biological marker in a biological sampleafter pulsing with an agent, in order to screen for a compound, saidcompound used for the production of a medicament for curing a disease.Therefore, the invention also relates to a compound identifiable by amethod according to present invention.

The devices, kits, and methods disclosed herein may be used to treatand/or diagnose diseases. An example of the disease to be cured ordiagnosed is an immuno-related disease. According to the invention,examples of immuno-related diseases may be autoimmunity, rheumatoidarthritis, multiple sclerosis, Type 1 diabetes mellitus, cancer (e.g. incancer immunotherapy), immunodeficiencies (e.g. in AIDS), allergy, graftrejection or Graft versus Host Disease (GVHD) (e.g. in transplantation).The examples enclosed in the present application illustrate saidapplications in detail. Therefore, a immunomodulatory compound or agentmay influence one of said diseases; the change of the immuno-relatedtranscripts or the epitope specific CTLs-related or T Helperlymphocyte-related transcripts may indicate the presence and/or thestatus of one of said diseases; as well as the immunological statuswhich may illustrate the status of one of said diseases.

Nucleic acids which may be quantified using the devices, kits, andmethods of the present invention in order to study said immuno-relateddisease may be nucleic acids coding for, for example, chemokines,cytokines, growth factors, cytotoxic markers, transcription factors,members of the TNF-related cytokine-receptor superfamily and theirligands, apoptosis markers, immunoglobulins, T-cell receptor, and anymarker related to the activation or the inhibition of the immune systemknown or to be discovered.

According to the invention, said nucleic acids may code for a markersuch as IL-1ra, IL-1β, IL-2, IL-4, IL-5, IL-9, IL-10, IL-12p35,IL-12p40, IL-13, TNF-α, IFN-γ, IFN-α, TGF-β, and any interleukin orcytokine involved or not in the immune response. House keeping genessuch β-actin or GAPDH (glyceraldehyde phosphate deshydrogenase) could beused as internal marker.

According to the invention said epitope specific CTLs-related or THelper lymphocyte-related transcripts be a nucleic acid coding forcytokines, cytokine receptors, cytotoxines, inflammatory oranti-inflammatory mediators, members of the TNF-relatedcytokine-receptor superfamily and their ligands, G-protein coupledreceptors and their ligands, tyrosine kinase receptors and theirligands, transcription factors, and proteins involved in intra-cellularsignaling pathways.

According to the present invention, said nucleic acid may code for amarker for any of granzyme, perforines, prostaglandins, leukotrienes,immunoglobulin and immunoglobulin superfamily receptors, Fas andFas-ligand, T cell receptor, chemokine and chemokine receptors,protein-tyrosine kinase C, protein-tyrosine kinase A, Signal Transducerand Activator of Transcription (STAT), NF-kB, T-bet, GATA-3, Oct-2.

The present invention also describes a use of a device, method or a kitaccording to the present invention, for thedetection/monitoring/screening of a compound, wherein said compound isan immunomodulatory compound which may be chosen from the groupconsisting of eukaryotic cells, prokaryotic cells, viruses, phages,parasites, drugs (natural extracts, organic molecule, peptide, proteins,nucleic acids), medical treatment, vaccine and transplants. The use ofsuch a method is not limited to detect/monitor/screen a single compound.Synergetic effects of group of substances can also be studied.

The present invention also relates to the use of any of the devices,kits, and methods as described above, for the detection/monitoring ofepitope specific CTLs or immuno-related transcripts.

The devices, kits, and methods according to the present invention canalso be applied for the monitoring of in vivo immunological responsesafter the treatment of patients with a drug/treatment/vaccinesusceptible to modify their immune status. According to the invention,the detection of cytokine mRNA (can be extended to chemokine, growthfactors, cytotoxic markers, apoptosis markers, or any marker relate tothe activation of the immune system known or to be discovered) with thedescribed method in whole blood of patients under therapy or enrolled inclinical trials with an immunomodulator drug or treatment or with avaccine (therapeutic or prophylactic) may be used to evaluate theefficiency, the safety and/or the eventual by-side effects of thetherapy.

The present invention also relates to a devices, kits, and methods forthe detection of in vivo immunological status for thediagnostic/prognostic of diseases affecting the immune system (cancer,auto-immune diseases, allergy, transplant rejection, GVHD, etc.)

According to the invention, the detection of cytokine mRNA (can beextended to chemokine, growth factors, cytotoxic markers, apoptosismarkers, or any marker relate to the activation of the immune systemknown or to be discovered) with the described method in whole blood ofpatients suffering a disease that affects directly of indirectly theirimmune system with the aim to dress a diagnosis or prognosis.

The present invention also describes a method to identify an agentcapable of modifying the immunological status of a subject via theanalysis of epitope specific CTLs comprising the steps of:

(a) applying an immunomodulatory agent(s) into a subject,

(b) sampling whole blood from said subject,

(c) pulsing blood cells present in the whole blood sample of step (b)with an identical/similar and/or different immunomodulatory agent asapplied in step (a), using a device as described above,

(d) collecting pulsed blood cells of step (c) or non-pulsed blood cellsof step (b) in a tube comprising a compound inhibiting RNA degradationand/or gene induction, or adding said compound to the pulsed/non-pulsedcells,

(e) forming a precipitate comprising nucleic acids,

(f) separating said precipitate of step (e) from the supernatant,

(g) dissolving said precipitate of step (f) using a buffer, forming asuspension,

(h) isolating nucleic acids from said suspension of step (g) using anautomated device,

(i) dispensing/distributing a reagent mix for RT-PCR using an automateddevice,

(j) dispensing/distributing the nucleic acids isolated in step (h)within the dispensed reagent mix of step (i) using an automated device,

(k) detecting/monitoring/analyzing the in vivo levels of epitopespecific CTLs-related transcripts in the dispensed solution of step (j)in an automated setup, and,

(l) identify agents able to modify the immunological status of saidsubject, whereby, in case the agent of step (a) is already present inthe subject, step (a) is omitted.

The present invention also relates to a kit comprising componentsenabling execution of at least step (c) above. The kit may containadditional reagents and instructions to enable one or more of the othersteps to be executed. The disclosures made herein instruct the skilledartisan of the components required to build the desired kit.

According to the present invention the immunomodulatory agent(s) may bepresent in case of a disease or in the presence of a transplant in saidsubject. In the present invention the ‘epitope specific CTLs-relatedtranscripts’ may be transcripts coding for cytokines, cytokinereceptors, cytotoxines (like granzyme, perforines, etc.), members of theTNF-related cytokine-receptor superfamily and their ligands (ex: Fas andFas-ligand) or other cellular receptors.

The present invention also describes a method to identify an agentcapable of modifying the immunological status of a subject:

(a) applying an immunomodulatory agent(s) into a subject,

(b) sampling whole blood from said subject,

(c) pulsing blood cells present in the whole blood sample of step (b)with an identical/similar and/or different immunomodulatory agent asapplied in step (a), using a device or kit as described above,

(d) collecting pulsed blood cells of step (c) or non-pulsed blood cellsof step (b) in a tube comprising a compound inhibiting RNA degradationand/or gene induction, or adding said compound to the pulsed/non-pulsedcells,

(e) forming a precipitate comprising nucleic acids,

(f) separating said precipitate of step (e) from the supernatant,

(g) dissolving said precipitate of step (f) using a buffer, forming asuspension,

(h) isolating nucleic acids from said suspension of step (g) using anautomated device,

(i) dispensing/distributing a reagent mix for RT-PCR using an automateddevice,

(j) dispensing/distributing the nucleic acids isolated in step (h)within the dispensed reagent mix of step (i) using an automated device,

(k) detecting/monitoring/analyzing the in vivo levels of immuno-relatedtranscripts in the dispensed solution of step (j) in an automated setup,and,

(l) identify agents able to modify the immunological status of saidsubject,

whereby, in case the agent of step (a) is already present in thesubject, step (a) is omitted.

The present invention also relates to a kit comprising componentsenabling execution of at least step (c) above. The kit may containadditional reagents and instructions to enable one or more of the othersteps to be executed. The disclosures made herein instruct the skilledartisan of the components required to build the desired kit.

In the present invention the ‘immuno-related transcripts’ may betranscripts coding for e.g. cytokine(s), chemokines(s), growth factors,cytotoxic markers, transcription factors, members of the TNF-relatedcytokine-receptor superfamily and their ligands, or any markers relatedto activation of the immune system known or to be discovered. Accordingto the present invention the immunomodulatory agent(s) may be present incase of a disease or in the presence of a transplant in said subject.The subject according to the present invention may be of both human oranimal origin.

The present invention also provides a method for thediagnosis/prognosis/monitoring of a clinical status affecting the immunesystem in a subject comprising the steps of:

(a) sampling whole blood from said subject,

(b) pulsing blood cells present in the whole blood sample of step (a)with an identical/similar and/or different immunomodulatory agent aspresent in the subject, using a device or kit as described above,

(c) collecting pulsed blood cells of step (b) in a tube comprising acompound inhibiting RNA degradation and/or gene induction, or addingsaid compound to the pulsed cells,

(d) forming a precipitate comprising nucleic acids,

(e) separating said precipitate of step (d) from the supernatant,

(f) dissolving said precipitate of step (e) using a buffer, forming asuspension,

(g) isolating nucleic acids from said suspension of step (f) using anautomated device,

(h) dispensing/distributing a reagent mix for RT-PCR using an automateddevice,

(i) dispensing/distributing the nucleic acids isolated in step (g)within the dispensed reagent mix of step (h) using an automated device,

(j) detecting/monitoring/analyzing the in vivo levels of immuno-relatedtranscripts in the dispensed solution of step (i) in an automated setup,and,

(k) detecting/monitoring the change in in vivo levels of immuno-relatedtranscripts, and,

(l) diagnosing/prognosing/monitoring the disease affecting the immunesystem.

In the present invention ‘clinical status’ is any change of the physicalcondition of a subject such as different diseases or presence oftransplants.

The present invention also relates to a kit comprising componentsenabling execution of at least step (c) above. The kit may containadditional reagents and instructions to enable one or more of the othersteps to be executed. The disclosures made herein instruct the skilledartisan of the components required to build the desired kit.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Exemplary methods and materialsare described below, although methods and materials similar orequivalent to those described herein can be used in the practice ortesting of the present invention. All publications and other referencesmentioned herein are incorporated by reference in their entirety. Incase of conflict, the present specification, including definitions, willcontrol. The materials, methods, and examples are illustrative only andnot intend to be limiting. Other features and advantages of theinvention will be apparent from the following figures, detaileddescription, and from the claims.

FIGURES

FIGS. 1 a to 1 d depict an example a vessel and method according to theinvention. FIG. 1 a shows a vessel 1 wherein antigen particles 2 arepresent. The vessel is fitted with a resealable means of entry 3 for asyringe needle. A container 4 is also part of the vessel 1; stabilisingagent 5 being present in the container, and the connection between theinside of the vessel 1 and the inside of the container 4 beingtemporarily blocked by a physical barrier, in this case, a plug 25. Ameans of transmitting physical force to dislodge the plug is provided inthe form of a plunger 28. In this example, the plunger is covered with acap 23. In FIG. 1 b, biological sample 24 is introduced into the vesselby way of a syringe needle 6, through the resealable means of entry 3,and the biological sample 24, is allowed to be exposed to the antigen 2.In FIG. 1 c, the plunger cap 23 is removed 26. In FIG. 1 d, a shaft 7 isintroduced and pressure applied thereto 27, so forcing the plunger 28 topush the plug 25 away from the container 4. Upon removal of the plug 25,stabilizing agent 5 is released into the vessel 1 and allowed to mixwith the biological sample 24 and antigen 2.

FIG. 2 depicts an example of a vessel 1 according to the invention inwhich antigen 2 is present. The vessel may be open-topped 7, as shownhere, or may be fitted with closures or means to introduce sample orstabilizing agent, examples of which are shown in FIGS. 3 to 6. The bodyof the vessel 1, may also comprise a container in which stabilisingagent is present as shown in FIGS. 7 and 8.

FIG. 3 depicts an example of a fitting suited for a type of vessel shownin FIG. 2. The top of the vessel 7, is fitted with a Luer-type fitting 8that can receive a reciprocal Luer type fitting on a syringe 11. Thesyringe may contain biological sample or stabilising agent according toembodiments of the invention.

FIG. 4 depicts an example of a fitting suited for a type of vessel shownin FIG. 2. The top of the vessel 7, is fitted with a resealable septum 9that can receive a syringe needle. The syringe may contain biologicalsample or stabilising agent according to embodiments of the invention.

FIG. 5 depicts an example of a fitting suited for a type of vessel shownin FIG. 2. The top of the vessel 7, is fitted a means to receive with ascrew cap 10.

FIG. 6 depicts an example of a fitting suited for a type of vessel shownin FIG. 2. The top of the vessel 7, is fitted a hypodermic syringeneedle 19. The vessel might be used directly to withdraw a sample froman individual.

FIG. 7 depicts an example of a body of a vessel 1 which could, forexample, be used in combination with the vessels and fittings shown inFIGS. 2 to 6. The vessel 1, in which antigen 2 is present, comprises acontainer 12 in which stabilizing agent 5 is present. The wall of thecontainer is made entirely or in part 15 from a material which shattersupon the application of a certain force. The container is fitted with ameans to transmit force from the user to shatter part or all of thecontainer, comprising a depressable area 13 attached to a sharp point14. Upon depression of the area 13, the sharp point 14 contacts theshatterable material 15, causing it to shatter, so removing the physicalbarrier between the container and the vessel, allowing the stabilisingagent to flow into the vessel 1 at a time determined by the user.

FIG. 8 depicts an example of a body of a vessel 1 which could, forexample, be used in combination with the vessels and fittings shown inFIGS. 2 to 6. The vessel 1, in which antigen 2 is present, comprises acontainer 16 in which stabilizing agent 5 is present. The connectionbetween the inside of the container and the vessel 17 is physicallyblocked by a septum 18 which is breachable by the application of force.Upon squeezing the wall of the container 16, pressure is transmitted tothe septum, causing the septum to breech so allowing the entry ofstabilising agent 5 into the vessel 1.

FIG. 9 depicts an example of a container 20 of the invention which isnot connected to a vessel. The stabilising agent 5 is present in thecontainer 20 and the container 20, is fitted with a Luer-type fitting 22suitable for coupling with a vessel having a reciprocal fitting (forexample as shown in FIG. 3 and FIG. 11). The walls of the container 20may be squeezable, allowing the stabilizing agent to exit upon theapplication of force thereto.

FIG. 10 depicts an example of a container 29 of the invention which isnot connected to a vessel. The stabilising agent 5 is present in thecontainer 29 and the container 29, is fitted with a Luer-type fitting 22for coupling with a vessel having a reciprocal fitting (for example asshown in FIG. 3 and FIG. 11). The vessel is further fitted with aplunger 30, the application of force on which allows the stabilizingagent 5 to exit.

FIG. 11 depicts an example of a kit according to the invention,comprising a vessel 1 fitted with a Luer type fitting 8 and, in thisinstance, a valve 31 allowing the exit of displaced air from vessel. Thekit also comprises a container 29, similar to that depicted in FIG. 10,in which stabilising agent 5 is present. The fitting on the vessel 8 iscapable of coupling to the fitting on the container 22.

FIGS. 12 a to 12 d depict an example a control vessel and methodaccording to the invention. FIG. 12 a shows a control vessel 37 whereincontrol substance 35 present. The control vessel is fitted with aresealable means of entry 3′ for a syringe needle. A control container4′ is also part of the control vessel 37; stabilising agent 5′ beingpresent in the control container, and the connection between the insideof the control vessel 37 and the inside of the control container 4′being temporarily blocked by a physical barrier, in this case, a plug25′. A means of transmitting physical force to dislodge the plug isprovided in the form of a plunger 28′. In this example, the plunger iscovered with a cap 23′. In FIG. 12 b, biological sample 24 is introducedinto the control vessel by way of a syringe needle 6′, through theresealable means of entry 3′, and the biological sample 24, is allowedto be exposed to the control substance 35. In FIG. 12 c, the plunger cap23′ is removed 26′. In FIG. 12 d, a shaft 7′ is introduced and pressureapplied thereto 27′, so forcing the plunger 28′ to push the plug 25′away from the control container 4′. Upon removal of the plug 25′,stabilizing agent 5′ is released into the control vessel 37 and allowedto mix with the biological sample 24 and control substance 35.

FIG. 13 depicts an example of a control vessel 37 according to theinvention in which a control substance 35 is present. The control vessel37 may be open-topped 7′, as shown here, or may be fitted with closuresor means to introduce sample or stabilizing agent, examples of which areshown in FIGS. 14 to 16. The body of the control vessel 37, may alsocomprise a control container 12′, 16′ in which stabilising agent ispresent as shown in FIGS. 17 and 18.

FIG. 14 depicts an example of a fitting suited for a type of controlvessel shown in FIG. 13. The top 7′ of the control vessel 35, is fittedwith a Luer-type fitting 8′ that can receive a reciprocal Luer typefitting on a syringe 11′. The syringe may contain biological sample orstabilising agent according to embodiments of the invention.

FIG. 15 depicts an example of a fitting suited for a type of controlvessel shown in FIG. 13. The top 7′ of the control vessel 37, is fittedwith a resealable septum 9′ that can receive a syringe needle. Thesyringe may contain biological sample or stabilising agent according toembodiments of the invention.

FIG. 16 depicts an example of a fitting suited for a type of controlvessel 37 shown in FIG. 37. The top 7′ of the control vessel 37, isfitted a means to receive with a screw cap 10′.

FIG. 17 depicts an example of a body of a control vessel 37 which could,for example, be used in combination with the control vessels andfittings shown in FIGS. 14 to 16. The control vessel 37, in whichcontrol substance 35 is present, comprises a control container 12′ inwhich stabilizing agent 5′ is present. The wall of the control containeris made entirely or in part 15′ from a material which shatters upon theapplication of a certain force. The control container is fitted with ameans to transmit force from the user to shatter part or all of thecontrol container, comprising a depressable area 13′ attached to a sharppoint 14′. Upon depression of the area 13′, the sharp point 14′ contactsthe shatterable material 15′, causing it to shatter, so removing thephysical barrier between the control container and the control vessel,allowing the stabilising agent to flow into the control vessel 37 at atime determined by the user.

FIG. 18 depicts an example of a body of a control vessel 37 which could,for example, be used in combination with the control vessels andfittings shown in FIGS. 14 to 16. The control vessel 37, in whichcontrol substance 35 is present, comprises a control container 16′ inwhich stabilizing agent 5 is present. The connection between the insideof the control container and the control vessel 37 is physically blockedby a septum 18′ which is breachable by the application of force. Uponsqueezing the wall of the control container 16′, pressure is transmittedto the septum, causing the septum to breech so allowing the entry ofstabilising agent 5 into the control vessel 37.

FIG. 19 depicts an example of a device of the invention comprising acontrol vessel 37 as described in FIG. 17, exteriorly connected to avessel 1 as described in FIG. 7 by way of a bridging member 70. Thedevice permits biological sample to be delivered separately to a firstsubstance 2 and control substance 35, prior to a stabilizing agent 5.Because the reaction and control samples are held side-by-side, errorsthat can arise with mixing control and reaction samples are avoided.

FIG. 20 depicts an example of a device of the invention comprising acontrol vessel 37 as described in FIG. 18, exteriorly mechanicallyconnected to a vessel 1 as described in FIG. 8 by way of a bridgingmember 70. The device permits delivery of biological sample separatelyto a first sample 2 and control substance 35, prior to a stabilizingagent 5. Because the reaction and control samples are held side-by-side,errors that can arise with mixing control and reaction samples areavoided.

FIG. 21 a shows a schematic illustration of an example of a device 30 ofthe invention for accepting a liquid biological sample, whichfacilitates exposure of said sample separately to a first substance anda control substance, and subsequently to a nucleic acid stabilizingagent, the device comprising. It comprise a first compartment 32 where afirst substance 2 is present, a second compartment 34 in which a controlsubstance 35 is present, a third compartment 36 where a stabilizingagent 5 is present, a support 52 for a biological sample tube that is acylindrical aperture. Each compartment is formed as an opening in ablock of solid material 44. Each compartment is sealed with a lid 46,48, 50 disposed with a resealable septum 38, 40, 42 that may be breechedwith a hollow needle such as a hypodermic cannular or needle. The deviceis further disposed with a support for a transfer tubing, which supporttakes the form of two slots 54, 56 adapted to accept the needle ends ofthe transfer tubing.

FIG. 21 b depicts a cross-section through a device of the inventionalong line A-A′, which shows in detail the contents of the second(control) compartment 34 and third (stabilizing agent) compartment 36.

FIG. 22 shows a cross-sectional view of a transfer tubing 60 of theinvention, comprising a hollow elongate tubing 64, whereby each end ofthe tubing is disposed with a hollow (e.g. cannular or hypodermic)needle 62, 66. The transfer tubing 60 is configured such that liquid canflow through one needle 62 along the tubing 64 to the other needle 66.In the embodiment shown, the needles 62, 66 are of unequal length.

FIG. 23 shows a cross-sectional view of a pressurising tubing 88 of theinvention, comprising a hollow flexible elongate tubing 82, whereby oneend of the tubing is in connection with a hollow (e.g. cannular orhypodermic) needle 80 and the other end 85 is adapted for connection theoutlet coupling 87 of a (pressurizing or evacuating) gaseous media (e.g.air) pump 86. The pressurising tubing 88 is configured to pierce theseptum of a vessel, or compartment, and create a differential pressuretherein by the evacuation or introduction of gaseous medium (e.g. air).

FIG. 24 shows a cross-sectional view of a combined set comprising thepressurising tubing 88 of the invention and the transfer tubing 60. Oneneedle 62 of the transfer tubing 60 is connected using one or morejoints 84 to the needle 80 of the pressurising tubing 88 such that bothneedles 62, 80 are in parallel alignment. Thus, a septum issimultaneously pierced by both needles 62, 80.

FIG. 25 shows a cross-sectional view of a transfer tubing 60 of theinvention, with dimensions indicated. D represents the minimum distancebetween two needles 62, 66. L1 is the length of one needle, particularlythe straight part, L2 is the length of the other needle particularly thestraight part, L3 is the distance on a first imaginary straight line 65,that extends from the straight part of a needle 62, 66 towards thetubing 64, which distance L3 is defined by end 67 of the straight partof the needle, and the point 69 where a second imaginary straight line63 crosses the first imaginary straight 65, which second imaginarystraight line 63 intersects the top of the tubing 64.

FIGS. 26 to 29 shows an example of the device 30 as shown in FIG. 21 ain use.

In FIG. 26 a biological sample tube 58 comprising liquid biologicalsample (e.g. blood, urine) is inserted into the tubing support 52. Oneend of the transferring tubing 60 is inserted into the biological sampletube 58 through the septum 61, and the other end is inserted into thesecond (control) compartment 34, through the septum 40. Liquidbiological sample flows from the biological sample tube 58, along thetransfer tubing 60 in the direction of the arrow, and into the secondcompartment 34, where it contacts the control substance 35. Liquidbiological sample may be propelled by the use of pressurizing tubing 88(not shown), the needle of which may pierce either the septum of the 38of the second compartment 34 and a vacuum created, or the septum 61 ofthe biological sample tube 58 and pressure therein increased.

In FIG. 27, the transfer tubing has been removed and reinserted, suchthat the end of transfer tubing 60 in the second compartment 34 ispositioned in the first compartment 32, through the septum 38, while theend of transfer tubing 60 in the biological sample tube 58 remainsthere. Liquid biological sample flows from the biological sample tube58, along the transfer tubing 60 in the direction of the arrow, and intothe first compartment 32, where it contacts the first substance 2(pulsing agent). Liquid biological sample may be propelled by the use ofpressurizing tubing 88 (not shown), the needle of which may pierceeither the septum of the 40 of the first compartment 32 and a vacuumcreated, or the septum 61 of the biological sample tube 58 and pressuretherein increased.

FIG. 28 shows rinsing of the transfer tubing 60 after transfer of liquidbiological sample, by removal and reinsertion of the transfer tubing 60such that the end of the transfer tubing 60 previously in contact withthe biological sample tube 58, is inserted into the third (stabilizingagent) compartment 36 through the septum 42, and the other end of thetransfer tubing 60 is inserted into the biological sample tube 58through the septum 61. Liquid stabilizing agent 5 flows from the thirdcompartment 36, along the transfer tubing 60 in the direction of thearrow, and into the biological sample tube 58, where it is stored aswaste or for further processing. Liquid stabilizing agent 5 may bepropelled by the use of pressurizing tubing 88 (not shown), the needleof which may pierce either the septum of the 42 of the third compartment36 and pressure therein increased, or the septum 61 of the biologicalsample tube 58 and a vacuum created. It is noted that after rinsing, thesamples are allowed to incubate. The period of incubation and thetemperature will depend on the several factors including the pulsingagent, the sample and the volume and concentration of reagents. Forgeneral guidance, incubation may be performed at 37 deg C. for between30 minutes and 16 hours, most typically between 2 and 4 hours.

In FIG. 29, the transfer tubing 60 is removed and reinserted, such thatone end is inserted into the third (stabilizing agent) compartment 36,through the septum 42, and the other end is inserted into the second(control) compartment 34 through the septum 40. Liquid stabilizing agent5 flows from the third compartment 36, along the transfer tubing 60 inthe direction of the arrow, and into the second compartment 34, where itcontacts the mixture of biological sample and control substance 2.Liquid stabilizing agent 5 may be propelled by the use of pressurizingtubing 88 (not shown), the needle of which may pierce either the septumof the 42 of the third compartment 36 and pressure therein increased, orthe septum 40 of the second compartment 34 and a vacuum created.

In FIG. 30, the transfer tubing 60 is removed and reinserted such thatone end is inserted into the third (stabilizing agent) compartment 36,through the septum 42, and the other end is inserted into the first(pulsing reaction) compartment 32, through the septum 38. Liquidstabilizing agent flows from the third compartment 36, along thetransfer tubing 60 in the direction of the arrow, and into the firstcompartment 32, where it contacts the mixture of biological sample andfirst substance 35. Liquid stabilizing agent 5 may be propelled by theuse of pressurizing tubing 88 (not shown), the needle of which maypierce either the septum of the 42 of the third compartment 36 andpressure therein increased, or the septum 38 of the first compartment 32and a vacuum created.

At the end of sequence, both the first and second compartments havereceived biological sample and stabilizing agent. The movements by thetransfer tubing 60 and pressurizing tubing 88 where used may be actuatedrobotically, e.g. by use of a robotic arm having several degree offreedom of movement or by an X-Y robotic table. When robotic actuationis employed, the transfer tubing will typically comprise a rigid tubing64 that permits the transfer tubing 60 to be gripped and positioned withaccuracy.

FIGS. 31.1 to 31.4 Strategies followed in the given examples

FIG. 31.1 Ex vivo monitoring of immune response against tetanus toxoid.

FIG. 31.2 Strategy followed in example 3.

FIG. 31.3 Strategy followed in example 4

FIG. 31.4 Strategy followed in example 5.

FIG. 32.1: RT-PCR for spontaneous production of IFN-γ, and IL-10 mRNAsin peripheral blood. Total RNA was extracted from whole blood and fromPBMC, as stated, from six different healthy volunteers (columns 1 to 6).Whole blood: 0.6 ml of whole blood were mixed with 6 ml of Catrimox-14™,within the minute that follows sample collection. The samples were thencentrifuged at 12000 g for 5 min. The resulting nucleic acids pellet wascarefully washed with water, and dissolved in 1 ml of Tripure™. RNAextraction was then carried out according to Tripure™ manufacturer'sinstructions. PBMC: cells were prepared following standard proceduresfrom 15 ml of heparinized venous blood, and lysed in 1 ml of Tripure™for RNA extraction. RT-PCR for IFN-γ, IL-10 and housekeeping gene HPRTwere performed for all samples from 1 μg total RNA as described(Stordeur et al., (1995), Pradier et al., (1996)).

FIG. 32.2: Real time PCR for IFN-γ and IL-10 mRNA stability in wholeblood. A sample of citrated venous blood was collected from healthydonors. From this sample, a 100 μl aliquot was mixed with 900 μl ofCatrimox-14™, within the minute that follows blood collection, and everyhour after during five hours, the blood sample being simply kept at roomtemperature between each aliquot taking. The resulting nucleic acidspellet (see legend to FIG. 13.1) was dissolved in 300 μl lysis bufferfrom the “MagNA Pure LC mRNA Isolation Kit I” (Roche Diagnostics,Molecular Biochemicals). mRNA was extracted using the MagNA Pure LCInstrument (Roche Diagnostics, Molecular Biochemicals) followingmanufacturer's instructions (final elution volume: 100 μl). Reversetranscription and real time PCR were performed in one step, followingthe standard procedure described in the “Lightcycler—RNA MasterHybridisation Probes Kit” (Roche Diagnostics, Molecular Biochemicals),starting from 5 μl of the mRNA preparation. Primers and probessequences, and PCR conditions, are described in Stordeur et al, JImmunol Methods, 259 (1-2): 55-64, 2002).

FIG. 33: Schematic comparison of the RNA extraction method from wholeblood as suggested by PreAnalytiX compared to method as proposed by thepresent invention.

FIG. 34. Cytokine blood mRNA ex vivo induction by tetanus toxoid.Tetanus toxoid (10 μg/ml, Aventis) was added to 500 μl whole bloodcollected from healthy volunteer vaccinated against tetanus seven yearsago. After different time periods at 37° C. in a 5% CO₂ atmosphere, 1.4ml of the reagent contained in the PAXgene tube was added. 300 μl of theobtained lysate were used to isolate total mRNA on the MagNA Pureinstrument, and RT-PCR was performed as described in the presentinvention.

FIG. 35. IL-1β and IL-1 RA mRNA kinetics after whole blood stimulationwith LPS. 200 μl of heparinized blood were incubated with 10 ng/ml LPSfor 0 (beginning of the culture), 0.5, 1, 2 and 6 hours. At the end ofthe culture, 500 μl of the PAXgene™ tube's reagent were added for totalcell lysis and nucleic acid precipitation. Then RT and real time PCR forIL-1β, IL-1RA and β-actin mRNAs were performed in one step as describedin the present invention. Results are expressed in mRNA copy numbers permillion of β-actin mRNA copies. The mean and standard error on the meanof five independent experiments are shown.

FIG. 36. Linear regression: mRNA copy numbers on starting blood volume.Various whole blood volumes (ranging from 20 to 200 μl, X-axis) werecultured in the presence of 10 ng/ml LPS for six hours. At the end ofthe culture, RT and real time PCR for IL-1β and β-actin mRNAs wereperformed as described in the present invention. The Y-axis representsthe raw copy numbers. The line is for linear regression. One experimentrepresentative of six is shown.

FIG. 37. mRNA cytokine kinetics after whole blood stimulation withtetanus toxoid. Heparinized blood has been taken from five healthyvolunteers who were vaccinated against tetanus at least five years ago.For each donor, 200 μl whole blood aliquots were incubated with 10 μg/mltetanus toxoid for 0 (beginning of the culture), 4, 8, 16, 24 and 48hours. At the end of the culture, 500 μl of the reagent contained in thePAXgene™ tube were added, and the different transcripts quantified usingthe methodology of the present invention. Results are expressed in mRNAcopy numbers per million of β-actin mRNA copies. The mean and standarderror on the mean of five independent experiments are shown.

FIG. 38. In vivo modulation of blood cytokine mRNAs after intravenousinjection of LPS. Five healthy volunteers were injected with a singledose of 4 ng/kg LPS. Ten minutes before, and 0.5, 1, 1.5, 2, 3 and 6hours after the LPS injection, a 2.5 ml sample of blood was taken in aPAXgene™ tube. Quantification of cytokine mRNAs was performed accordingto the method of the present invention. Results are expressed in mRNAcopy numbers per million of β-actin mRNA copies. The mean and standarderror on the mean for each time point are represented.

FIG. 39. Follow-up of anti-tetanus vaccine response. Six healthyvolunteers were selected to receive an anti-tetanus recall. IL-2 mRNAlevels were quantified from whole blood cultured for 20 hours with (fullcircles) or without (open circles) 10 μg/ml tetanus toxoid, andperformed at the moment of the recall (day 0), 14 days before, and 3, 7,14, 21 and 90 days after (X-axis). Results are expressed in mRNA copynumbers per million of. β-actin mRNA copies (Y-axis). Each of the sixpanels (numbered 1 to 6) represents individual data from 6 differentdonors (one donor per panel).

FIG. 40. Summary of the procedure followed in examples 7, 8, 9, 10 and11 for analysing blood cytokine mRNA expression.

FIG. 41. Automated mRNA extraction and reagent mix preparation on theMagNA Pure: direct correlation between amount of starting biologicalmaterial and found copy number. The Y-axis represents the raw copynumbers. The line is for linear regression.

FIG. 42. Automated mRNA extraction and reagent mix preparation on theMagNA Pure. direct correlation between amount of starting biologicalmaterial and found copy number The Y-axis represents the raw copynumbers. The line is for linear regression.

FIG. 43. Summarised case report of the patient enrolled for cancerimmunotherapy. The melanoma was diagnosed in July 1999. In Augusts 2001,multiple metastasis were evidenced, and directly after an orchydectomyin April 2002, the patient was enrolled for receiving a cancer vaccine.The vaccine consisted in several injections of the MAGE-3 purifiedprotein (an antigen specifically expressed by melanoma cells) incombination with an adjuvant.

FIG. 44. Schematic representation of the vaccination protocol and themonitoring of immune response by real-time PCR. Whole blood wasstimulated in vitro to assess immune response to MAGE-3 The patientreceived 3 injections of the vaccine, while a blood sample was takenonce a week during 9 weeks. A 200 μl aliquot of each patient's wholeblood sample was incubated in the presence of 10 μg/ml MAGE-3 protein or10 μg/ml TRAP (plasmodium falciparum antigen) as a negative control. Atthe end of the culture, the reagent contained in the PAXgene tube wasadded to allow IL-2 mRNA quantification as described in example 6. Theresults are presented in FIG. 45.

FIG. 45. IL-2 mRNA in whole blood following MAGE-3 vaccination inPatient #3. Higher IL-2 mRNA levels are observed in MAGE-3-stimulatedwhole blood after MAGE-3 vaccine boost. The Y-axis represents the IL-2mRNA copy numbers per million of β-actin mRNA copies, and the X-axis theweeks at which blood samples were taken. The vaccine injections wereadministrated at the weeks 0, 2 and 6. The solid columns are for wholeblood incubated in the presence of MAGE-3, and the hatched columns forwhole blood incubated in the presence of TRAP.

FIG. 46. In vitro stimulation of whole blood: evaluation of immuneresponse to allergen. The experiment was performed for IL-4 mRNAquantification after whole blood incubation with an allergen. Bloodsamples were taken from a subject allergic to cat, and from two healthysubjects. Whole blood was then incubated in absence or in the presenceof the cat allergen (namely Feld1), for different time periods ofculture, at the end of which the reagent contained in the PAXgene tubewas added to allow IL-4 mRNA quantification as described in example 6.The results are presented on FIG. 47.

FIG. 47. In vitro stimulation of whole blood: evaluation of immuneresponse to Feld1. Feld1 allergen significantly induces higher IL-4 mRNAlevels in whole blood coming from the subject allergic to the catcompared to non allergic subjects. The Y-axis represents the IL-4 mRNAcopy numbers per million of β-actin mRNA copies, and the X-axis thedifferent incubation times. Green columns represent IL-4 mRNA levelsfound in normal whole blood incubated with the allergen, IL-4 mRNAlevels found in whole blood of the allergic subject being represented bythe red columns (blood incubated in the presence of Feld1) and theyellow columns (blood incubated without Feld1).

FIG. 48. The response to Feld1 in this whole blood system is specificand dose-related. Whole blood from the allergic subject was incubatedfor two hours 1) in the presence of increasing concentrations of Feld1(diagonal hatched columns); 2) in the presence of another allergen,β-lactoglobulin (BLG) at 10 μg/ml (horizontal broken hatched column); 3)crossed-linked IgE (dotted column). The Y-axis represents the IL-4 mRNAcopy numbers per million of β-actin mRNA copies.

FIG. 49. IL-4 mRNA levels after whole blood stimulation with Feld1 arehigher in patients allergic to the cat compared to healthy controls. Theexperiment described on slides 9 to 11 was repeated on blood samplesfrom 10 healthy subjects (CTR columns) and 10 patients allergic to thecat (ALL columns). Whole blood samples were incubated for two hours inthe presence of 10 μg Feld1, or in the presence of crossed-linked IgE aspositive controls. The mean and standard error on the mean arerepresented.

FIG. 50. In vitro stimulation of whole blood: assessment of T cellresponse to GAD65. Schematic representation of the experiment performedfor IL-2 mRNA quantification after whole blood incubation with purifiedGAD65 protein. Blood samples were taken from six type 1 diabetespatients, and from five healthy subjects. Whole blood was then incubatedwithout or with 10 μg/ml GAD65 for 18 hours, the culture being thenstopped by adding the reagent contained in the PAXgene tube. IL-2 mRNAlevels were then quantified as described in example 6. The results arepresented in FIG. 51.

FIG. 51. In vitro stimulation of whole blood: assessment of T cellresponse to GAD65. Whole blood from type 1 diabetes patients showshigher IL-2 mRNA levels after GAD65 stimulation compared to healthysubjects. Results are expressed in IL-2 mRNA copy numbers calculatedrelatively to the copy numbers found in whole blood cultured withoutGAD65, after correction against β-actin. A logarithmic scale is used.The mean and standard error on the mean are represented. Healthy donors:CTR column; autoimmune diabetes patients: PAT column.

FIG. 52. Monitoring of alloreactive immune response: quantification ofIL-2 mRNA in a whole blood+dendritic cells system. Experiment performedfor IL-2 mRNA quantification after whole blood incubation with unrelateddendritic cells (DC) to assess alloreactive T cell response. Dendriticcells from two unrelated healthy volunteers (MT and MA) were generatedin vitro in the presence of IL-4 and GM-CSF. A whole blood sample fromeach donor was cultured in the presence of the dendritic cell populationof the other donor (1) or in the presence of their own dendritic cells(2). Whole blood samples from both donors were mixed (3), as well asboth dendritic cell preparations (4). After 12 hours incubation, thecultures were stopped by adding the reagent contained in the PAXgenetube. IL-2 mRNA levels were then quantified as described in example 6.The results are shown in FIG. 53.

FIG. 53. Monitoring of alloreactive immune response: quantification ofIL-2 mRNA in a whole blood+dendritic cells system. Assessment ofalloreactive T cell response by IL-2 mRNA quantification in whole blood.IL-2 mRNA copy numbers per million of β-actin mRNA copies are shown. Theconditions are, from left to right: whole blood from donor MA alone,whole blood from donor MA+DC from donor MA, whole blood from donor MA+DCfrom donor MT, whole blood from donor MT alone, whole blood from donorMT+DC from donor MT, whole blood from donor MT+DC from donor MA, wholeblood from donor MT+whole blood from donor MA, DC from donor MT+DC fromdonor MA.

EXAMPLES Example 1 Analysis of Spontaneous Cytokine mRNA Production inPeripheral Blood

The quantification of the cytokine mRNAs synthesized by peripheral bloodcells should make it possible to estimate a “peripheral immune statute”.However, an accurate quantification can only be performed from a freshwhole blood sample in which mRNA is protected against nucleasedigestion, and where gene transcription is inhibited. As discussed inthis note, this has been made possible by the use of surfactant reagentssuch as tetradecyltrimethylammonium oxalate. RT-PCR for thequantification of IL-10 and IFN-γ mRNAs spontaneously produced inperipheral blood was performed. The results showed pronounced higherIFN-γ transcript levels in whole blood compared to peripheral bloodmononuclear cells (PBMC) from the same individuals, while no significantdifference was observed for IL-10 mRNA. The higher amounts of IFN-γ mRNAobserved in blood can be attributed at least to mRNA degradation. Usinga real time PCR technique, it could indeed be demonstrated that bloodIFN-γ mRNA is rapidly degraded in vitro, the t ½ being worthapproximately one hour at room temperature.

Härtel et al. recently analysed the influence of cell purificationprocedure on spontaneous cytokine mRNA production in peripheral blood(Hartel et al., 2001). They showed that freshly isolated peripheralblood mononuclear cells (PBMC) expressed higher levels of IL-2, IL-4 andTNF-α mRNA than freshly collected whole blood from the same individual,while no difference in IFN-γ mRNA level was observed. A comparison forIFN-γ in six different individuals was performed, and different resultswere found. A strong expression of IFN-γ mRNA in whole blood of alldonors was observed, which is clearly decreased in PBMC (FIG. 32.1).This difference between the results obtained and those of Härtel et al,despite the fact that these latter used a quantitative real time PCRtechnique, could be related to the procedure used to isolate total RNAfrom whole blood. Härtel et al. used heparinized blood that washemolyzed within two hours by isotonic ammonium chloride treatment. Inthe present method tetradecyltrimethylammonium oxalate was used, acationic surfactant reagent called Catrimox-14™ (Qiagen, Westburg,Leusden, The Netherlands) that is directly mixed with the blood,avoiding the use of anticoagulants (Dahle and Macfarlane, (1993);Schmidt et al., (1995)). Moreover, this reagent induces nucleic acidsprecipitation and nuclease inhibition, in the minute that follows samplecollection. This provides a total RNA preparation that is probably thenearest of in vivo mRNA status. This is especially important forcytokine mRNA, which are made sensitive to endogenous nucleases by theirAU-rich sequences located in their 3′ untranslated region. Using a realtime PCR technique, it was indeed observed that peripheral blood IFN-γmRNA is spontaneously and rapidly degraded, the levels being decreasedby roughly 50% already one hour after blood collection. However, thisphenomenon is not necessary true for all the cytokines, as it was foundthat IL-10 mRNA level is stable for at least the five hours that followblood sampling (FIG. 32.2). Moreover, no significant differences inwhole blood IL-10 mRNA levels were found, compared to those of PBMC(FIG. 32.1).

The nucleic acids pellet obtained after Catrimox-14™ lysis (see legendto FIG. 32.1) can be dissolved in the guanidium/thiocyanate solutiondescribed by Chomczynski and Sacchi (1987), as well as in itscommercially available version, such as Tripure™ Roche Diagnostics,Molecular Biochemicals, Brussels, Belgium), making the use of thissurfactant particularly easy. This means that, except for the first stepwith Catrimox-14™, the RNA isolation procedure is the same for wholeblood and cells. Alternatively, PAXgene™ Blood RNA Tubes (Qiagen,Westburg, Leusden, The Netherlands) could be used in the place ofCatrimox-14™. In this case, the resulting pellet can be dissolved in thelysis buffer of the “MagNA Pure LC mRNA Isolation Kit I”, as describedfor Catrimox-14™ in legend to FIG. 32.2. The characterisation ofspontaneous IL-10 mRNA production by human mononuclear blood cells(Stordeur et al., (1995)), and the monitoring of in vivo tissue factormRNA induction by OKT3 monoclonal antibody (Pradier et al., (1996)),represent two examples where Catrimox-14 was successfully used. A strongIL-2 mRNA induction was also observed after addition of ionophoreA23187+phorbol myristate acetate to whole blood (not shown), suggestingits use for in vitro studies on whole blood.

The observations made in the present example stress the importance toperform RT-PCR from whole blood lysed as fast as possible, in order toaccurately quantify peripheral blood cytokine mRNA. For this purpose,the use of reagents such as Catrimox-14 or the additive contained in thePAXgene™ Blood RNA Tubes, together with real time RT-PCR, probablyrepresents to-date the best procedure. By doing so, the study of thenatural status of peripheral blood cells would be possible without theuse of in vitro strong stimuli such as ionomycin or phytohaemagglutinin.

Example 2 Comparison Between the PAXgene™ Blood RNA System and ProposedMethod According to the Present Invention

With the ‘PAXgene™ Blood RNA System’ is meant the combination of thePAXgene™ Blood RNA Tube’ with the ‘PAXgene™ Blood RNA Kit’. With the‘Qiagen Method’, it is meant ‘PAXgene™ Blood RNA Kit’.

Based on the experimental evidence described in Stordeur et al, JImmunol Methods, 259 (1-2): 55-64, 2002, the present invention proposesa new procedure to isolate mRNA from whole blood which allows todetermine in vivo transcript levels using an easy and reproduciblemethod. The PAXgene™ blood RNA System and the method according topresent invention are schematically compared in FIG. 33.

Material and Methods:

All experiments were performed from peripheral venous blood directlycollected in PAXgene™ Blood RNA Tubes as recommended by the PAXgene™Blood RNA System (Qiagen) (i.e. 2.5 ml of blood were vacuum collectedwithin the tube that contains 6.9 ml of an unknown reagent). After lysiscompletion, the content of the tube was transferred in two other tubes:4.7 ml were used for PAXgene blood RNA kit, and 0.4 ml for MagNA Pureextraction. The remaining of the lysate was discarded. These two tubeswere centrifuged at 2,000 g for 10 min and the supernatant discarded.The nucleic acid pellet was then:

a) PAXgene™ Blood RNA Tube+PAXgene™ Blood RNA Kit— . . . washed in waterbefore being dissolved in BR1 buffer for total RNA extraction, asrecommended in the corresponding instruction manual. The procedure ofthe PAXgene™ Blood RNA System is as follows: Blood samples (2.5 ml) arecollected in PAXgene Blood RNA Tubes, and may be stored or transportedat room temperature if desired. RNA isolation begins with acentrifugation step to pellet nucleic acids in the PAXgene Blood RNATube. The pellet is washed, and Proteinase K is added to bring aboutprotein digestion. Alcohol is added to adjust binding conditions, andthe sample is applied to a spin column as provided by the PAXgene™ BloodRNA Kit. During a brief centrifugation, RNA is selectively bound to thesilica-gel membrane as provided by the PAXgene™ Blood RNA Kit ascontaminants pass through. Following washing steps, RNA is eluted in anoptimized buffer. Reverse transcription and real time PCR for IFN-γ andβ-actin mRNAs were conducted as described by Stordeur et al. (“CytokinemRNA Quantification by Real Time PCR” J Immunol Methods, 259 (1-2):55-64, 2002).b) PAXgene™ Blood RNA Tube++MagNA Pure LC mRNA Isolation Kit I— . . .dissolved in 300 μl lysis buffer from the MagNA Pure mRNA Isolation Kit.Extraction and purification of mRNA in a final elution volume of 100 μlwere then performed on the MagNA Pure LC Instrument following theinstructions from Roche Diagnostics, Molecular Biochemicals, Reversetranscription and real time PCR were conducted in one step, followingthe standard procedure described in the “Lightcycler—RNA MasterHybridisation Probes Kit” (Roche Diagnostics, Molecular Biochemicals),starting from 5 μl of the mRNA preparation.Results:

A comparison of the extraction method recommended by Qiagen incombination with the PAXgene™ Blood RNA Tubes (PAXgene™ Blood RNASystem), with the MagNA Pure LC Instrument extraction method also incombination with the PAXgene™ Blood RNA Tubes was performed. In bothmethods the use of the PAXgene™ blood RNA Tubes allows to stabilize RNAfrom blood cells. The results are listed in Table 1.1 and 1.2. Theresults of this experiment show a better reproducibility for the MagNAPure LC Technique (coefficients of variation for IFN-γ mRNA copy numberscorrected against β-actin are 26 versus 16% for Qiagen versus MagNA PureLC, respectively).

It is interesting to note that MagNA Pure extraction was performed froma starting blood volume lower than that used with the Qiagen method(0.11 ml for MagNA Pure versus 1.25 ml for Qiagen). If the Qiagen methodhad been performed with such small volume, it would be impossible tomeasure the RNA concentration, even to perform the reversetranscription. This stresses another advantage of the techniquedescribed in the present invention: the possibility to quantify mRNA ina very small volume of blood (about 100 μl).

Conclusion

Example 2 illustrates the possibility to use the PAXgene™ Blood RNATubes in combination with the MagNA Pure LC mRNA Isolation Kit I, ormore precisely, the possibility to dissolve the precipitate from thePAXgene™ Blood RNA Tube in the lysis buffer contained in that kit, thislysis buffer necessarily having to be used with the other components ofthe kit.

In this example it is proven that in contrast to other combinations,only the combination as described in the present invention, leads tocorrect/real in vivo transcript quantification.

Example 3 Ex Vivo Monitoring of Immune Response Against Tetanus Toxoid

In example 3, blood is stimulated ex vivo with an antigen (i.e. tetanustoxoid) against which the blood donor is supposed to be immunised(because vaccinated seven years ago). RT-PCR is performed according tothe method (FIG. 31.1). Cytokine mRNA is measured as a read out of theability of the volunteer's immune system to react against the antigen.The IL-2, IL-4, IL-13 and IFN-γ mRNAs are preferentially analysed, butall potentially reactive proteins can be analysed via the quantificationof their corresponding mRNA. Results of example 3 is shown in FIG. 34.Generally the strategy followed in this example can be schematicallyrepresented as shown in FIG. 31.2.

Example of Possible Application Cancer Immunotherapy

Since some years, basic strategies on cancer immunotherapy evolved inthe way of the vaccination. In fact, the progresses in genetic and inimmunology have allowed identifying a number growing tumor antigens thatare expressed to the surface of tumor cells. These antigens arepresented to the surface of tumor cells under the form of peptidesassociated to the major histocompatibility complex (HLA). Example ofantigens that might be considered as tumor antigens are described byFong and Engleman (Annu. Rev. Immunol. 2000. 18:245-273). The principleof the anti-cancer vaccination consists to present these antigens to thesystem immune of the patient following the most immunogenic wayimmunogenic. That goes from the injection of the antigen orcorresponding peptides in the presence of additives to the presentationof the peptide on autologous antigen presenting cells (dendritic cells,for example). Although the ultimate goal of vaccination anti-cancervaccination remains the regression of the tumor, the determination ofthe efficiency of anti-cancer vaccination remains difficult especiallyin the case of patients in advanced phase of the disease that can profitonly from a limited window of treatment. It is the reason why theanti-cancer vaccination could especially be interesting as adjuvanttherapy or in the framework of the prevention. It is therefore extremelyimportant to develop sensitive and precise monitoring techniques toevaluate the immunological effects of the experimental anti-cancervaccination in order to specify the method of administration of thesevaccines and discover the implied biological mechanisms that will beable to help better to define the futures therapeutic protocols. Thedifficulty to measure the immunological efficiency of these vaccinesresides essentially in the absence of assays sufficiently sensitive todetect a cellular immune response in vivo. Until now, the usedtechniques implied the intensive in vitro culture of the PBMC ofpatients on of long periods times in the presence of antigen and ofco-stimulating susceptible to induce a modification of the originalfunctional characteristics of lymphocytes. Thus, the analyses of theanergic states or tolerant states of the lymphocyte precursors directedagainst the tumor antigens is extremely difficult being given thereversible nature of their functional state after their extendedin-vitro incubation in the presence of antigen. On the other side,techniques based on tetramers of MHC-peptides complexes that are usedfor the detection of low frequencies of epitope-specific-CTL precursorslack usually sensitiveness for the detection of tumor-specificlymphocytes. In addition these techniques do not give any information onthe functional reactivity of these lymphocytes

Only techniques that are sensitive enough to be able to detect anoriginal functional reactivity of the lymphocytes to a given antigen,for example after a very short stimulation in vitro with antigen willallow a real evaluation of the efficiency of anti-cancer vaccinationprotocols.

It has been shown recently (Kammula, U. S., Marincola, F. M., andRosenberg, S. A. (2000) Real-time quantitative polymerase chain reactionassessment of immune reactivity in melanoma patients after tumor peptidevaccination. J. Natl. Cancer Inst. 92: 1336-44) that the detection ofcytokine mRNA associated to a short in-vitro stimulation (2 hours) ofPBMC were able to detect epitope-specifiq CTLs in the PBMC's of patientsundergoing vaccination with a tumor antigen. Nevertheless, according tothe present invention this short ex vivo pulse is not essential.

Example 4 Detection of the Activation of the Immune System of theRecipient by the Histocompatibility Antigens of the Donor

In example 4, an organ (ex. liver, kidney, bone marrow, etc.) from adonor is transplanted to a recipient. Whole blood the recipient iscollected in a tube comprising a compound inhibiting RNA degradationand/or gene induction according to present invention. RT-PCR isperformed according to the method. Cytokine mRNA is measured as a readout of the activation of the immune system of the recipient by thehistocompatibility antigens of the donor (FIG. 31.3).

Example 5 Detection of the Reactivity of the Immune System of theRecipient to the Histocompatibility Antigens of the Donor

In example 5, an organ (ex. liver, kidney, bone marrow, . . . ) from adonor is transplanted to a recipient. Whole blood of the recipient iscollected on a tube and incubated ex-vivo with the histocompatibilityantigens of the donor. A compound inhibiting RNA degradation and/or geneinduction according to present invention is added to the blood. RT-PCRis performed according to the method. Cytokine mRNA is measured as aread out of the response of the immune system of the recipient by thehistocompatibility antigens of the donor (FIG. 31.4).

Example of Application Monitoring of Rejection after OrganTransplantation

The monitoring of rejections of transplants is essentially based on thedetection of markers measured in the urine or the blood of patients(blood urea nitrogen-BIN- or creatinine in the case of kidneytransplants) or at the time of the analyses of biopsies of the graftedorgan. These indicators are however only detected when the rejectionmechanism is already well advanced. In fact, transplant rejection is theresult of an immunological mechanism that precedes the deterioration ofthe grafted organ. The detection of these immunological mechanismsbefore the grafted organ is damaged would allow to reduce in aconsiderable manner the loss of the grafted organ by adapting moreearlier the immunosuppressive treatments. On the other side, it is alsorecognized that of sub-clinical episodes of rejections (with noinduction of clinical signs) occur themselves frequently aftertransplantation. These episodes sub-clinical rejection episodes could bethe cause of chronic rejections. Several authors have investigate thedetection of precocious immunologiques markers of organ rejection andparticularly the detection in the circulation of recipient alloreactiveT-lymphocytes directed against the allo-antigens of the donor. Methodsinclude essentially the association of mixed cultures with theconsecutive measurement of the proliferation of the lymphocytes of thereceiver or the measurement of the production of cytokines by differentmethods (ELISA, ELISPOT, flow cytometry, etc.). More recently, otherauthors have looked on the characterization of lymphocytes activationmarkers patterns susceptible to underline precociously the triggering ofa rejection mechanism. The detection of mRNA of genes expressed by thecytotoxic activated T-lymphocytes T activated (granzyme B, perforine,different cytokines) by sensitive methods of quantitative PCR wereshowed to be excellent tools to measure the triggering of a rejection.For this purpose, according to present invention, messengers coding fordifferent kinds of cytokines may be studied, preferential targets may beIL-2, IFN-gamma, IL-4, IL-5, Granzyme, perforine and FasFas-ligand.

Example 6 Immune Monitoring in Whole Blood Using Real Time PCR

In example 6 a whole blood method is described allowing the measure ofthe induction of cytokine synthesis at the mRNA level. The originalityof this method consists in the combination of PAXgene™ tubes containinga mRNA stabilizer for blood collection, the MagNA Pure™ instrument as anautomated system for mRNA extraction and RT-PCR reagent mix preparation,and the real time PCR methodology on the Lightcycler™ for accurate andreproducible quantification of transcript levels. This example firstdemonstrate that this method is adequate to measure the induction of IL(interleukin)-1β and IL-1 receptor antagonist (IL-1 RA) mRNA uponaddition of bacterial lipopolysaccharide (LPS) to whole blood. Thisexample further demonstrates that this approach is also suitable todetect the production of mRNA encoding T cell-derived cytokines in wholeblood incubated with tetanus toxoid as a model of in vitro immuneresponse to a recall antigen. Finally, the example demonstrates thatthis methodology can be used successfully to assess inflammatory as wellas T cell responses in vivo, as it allowed to detect the induction ofIL-1β and IL-1 RA after injection of LPS in healthy volunteers, and alsothe induction of IL-2 upon recall immunisation with tetanus vaccine.

Material and Methods.

Blood collection for in vivo studies. For accurate quantification ofperipheral blood mRNA levels, a 2.5-ml sample of blood was taken in aPAXgene™ tube for immediate cell lysis and nucleic acid precipitation.The mRNA is stable for up to 5 days in this blood lysate, the tubesbeing kept at room temperature until mRNA extraction.

In vitro whole blood culture. In vitro whole blood LPS stimulation ortetanus toxoid rechallenge were performed on 200 μl of heparinized wholeblood, and started at the latest four hours after blood collection.Cultures were stopped by adding 500 μl of the PAXgene™ tube's reagent,which induces total cell lysis and mRNA stabilisation. This allowed theuse of the same mRNA extraction protocol for both in vitro and in vivostudies.

mRNA extraction. The blood lysate obtained in the PAXgene™ tube or atthe end of whole blood culture was briefly mixed before transferring a300-μl aliquot in a 1.5-ml eppendorf tube for centrifugation at maximalspeed for 5 minutes (12,000 to 16,000 g, depending on the device). Thesupernatant was discarded, and the nucleic acid pellet thoroughlydissolved by vortexing in 300 μl of the lysis buffer contained in theMagNA Pure™ mRNA extraction kit (Roche Applied Science). mRNA was thenextracted from 300 μl of this solution, using this kit on the MagNAPure™ instrument (Roche Applied Science) following manufacturer'sinstructions (“mRNA I cells” Roche's protocol, final elution volume 100μl). The quality of the extracted mRNA was previously documented byNorthern blot analysis (Roche Applied Science, unpublished data).

Real time PCR and reagent mix preparation. Reverse transcription andreal time PCR were performed in one step, following the standardprocedure described in the “Lightcycler™-RNA Master HybridisationProbes” Kit (Roche Applied Science). More precisely, the RT-PCR reactionwas carried out in a 20 μl final volume containing: 1) H₂O up to 20 μl;2) 7.5 μl RNA Master Hybridisation Probes 2.7× conc (RNA MasterHybridisation Probes Kit—Roche Applied Science); 3) 1.3 μl 50 mM Mn(OAc)₂; 4) 1, 2 or 3 μl of 6 pmoles/μl forward and reverse primers(final concentration 300, 600 or 900 nM, depending of the mRNA target;the conditions specific for each mRNA target are fully described inStordeur et al, J Immunol Methods, 259 (1-2): 55-64, 2002, excepted forIL-2 and IL-4, which are listed in Table 2); 5) 1 μl of 4 pmoles/μlTaqMan probe (final concentration 200 nM); 6) 5 μl purified mRNA orstandard dilution. After an incubation period of 20 minutes at 61° C. toallow mRNA reverse transcription, and then an initial denaturation stepat 95° C. for 30 s, temperature cycling was initiated. Each cycleconsisted of 95° C. for 0 (zero) second and 60° C. for 20 s, thefluorescence being read at the end of this second step (F1/F2 channels,no colour compensation). 45 cycles were performed, in total. All primerswere chosen to span intronic sequences, so that genomic DNAamplification was not possible.

The RT-PCR reaction mixtures containing all reagents, oligonucleotidesand samples, were fully prepared directly in the capillaries used on theLightcycler™, by the MagNA Pure™ instrument. These capillaries were topclosed, centrifuged and then introduced in the Lightcycler™ for one stepRT-PCR. The sampling of all RT-PCR components was thus fully automated,avoiding manual sampling errors.

Results were expressed in copy numbers normalised against β-actin mRNA(mRNA copy numbers of cytokine mRNA per million of β-actin mRNA copies).For each sample, the mRNA copy number was calculated by the instrumentsoftware using the Ct value (“Arithmetic Fit point analysis”) from astandard curve. This latter was constructed for each PCR run from serialdilutions of a purified DNA, as described in Stordeur et al, J ImmunolMethods, 259 (1-2): 55-64, 2002.

Experimental endotoxemia. Five healthy male volunteers (21-28 years) whohad not taken any drugs for at least 10 days before the experiments werereceived an intravenous injection with a single dose of LPS (from E.coli, lot G; United States Pharmacopeial Convention, Rockville, Md.; 4ng/kg body weight). Ten minutes before, and 0.5, 1, 1.5, 2, 3 and 6hours after the LPS injection, a 2.5 ml sample of blood was taken in aPAXgene™ tube. For in vitro studies, 200 μl of heparinized whole bloodtaken from healthy individuals were incubated with 10 ng/ml LPS (from E.coli serotype 0128:B12, Sigma-Aldrich, Bornem, Belgium) for 0 (beginningof the culture), 0.5, 1, 2 and 6 hours, at 37° C. in a 5% CO₂atmosphere.

Anti tetanus recall vaccination. Healthy volunteers (2 males, 4 females,27-53 years) whom last tetanus toxoid vaccination was at least fiveyears ago, received an intra muscular vaccine recall (Tevax, Smith KlineBeecham Biologicals, Rixensart, Belgium). A heparinized blood tube wastaken the day of administration, 14 days before, and 3, 7, 14, 21 and 90days after. 200 μl of blood were incubated, at 37° C. in a 5% CO₂atmosphere, with or without 10 μg/ml tetanus toxoid (generous gift fromDr. E. Trannoy, Aventis Pasteur, Lyon, France) for 20 hours.

Results

Measurement of IL-1β and IL-1 RA mRNA upon addition of bacterial LPS towhole blood. As demonstrated in FIG. 35, addition of LPS (10 ng/ml) towhole blood led to a rapid induction of IL-1β and IL-1 RA mRNAs. Thisinduction, already evident 30 to 60 minutes after LPS addition, resulted6 hours after in a 47-fold and a 22-fold increase of the mRNA levels forIL-1β and IL-1 RA, respectively. The pattern of the curves suggests arapid and sustained increase of both cytokine mRNAs amounts. In order toevaluate the accuracy of the system for mRNA quantification, the mRNAwas quantified for β-actin and IL-1β from different volumes ofLPS-stimulated whole blood, ranging from 20 to 200 μl. As shown in FIG.36, the mRNA copy numbers of both β-actin and IL-1β were indeed directlycorrelated with the starting volume of blood.

In vitro response to tetanus toxoid. To determine whether this methodmight be suitable for the analysis of T cell responses, cytokine mRNAlevels in whole blood culture after addition of tetanus toxoid, a wellestablished recall antigen as all individuals were vaccinated inchildhood, was quantified. A rapid and transient induction of IFN-γ,IL-2, IL-4 and IL-13 mRNA after incubation of whole blood with thisantigen was found (FIG. 37). When comparing the amplitude of theresponse for each cytokine, it appeared that the induction of IL-2 mRNAwas the most pronounced. Indeed, the global increase of IL-2 mRNA copiesafter 16 hours of incubation in the presence of the toxoid was around220 fold for the five independent experiments shown in FIG. 37, whilethe maximum increase of IL-4 and IFN-γ mRNAs in the same experiments didnot exceed 5 fold. Quantification of IL-2 mRNA therefore appears as themost sensitive parameters in this whole blood system assessing T cellresponses. Data given in Table 3 indicates that the amplitude of theresponse to tetanus toxoid in this test is rather variable, probablydepending on the moment of the last vaccine recall. The induction ofIL-2 mRNA was effectively not observed after addition of tetanus toxoidto neonatal cord blood, indicating that only previously primed T cellsand not naive T cells are able to respond in this assay (Table 3).

Induction of IL-1 RA and IL-1β mRNA in whole blood after intravenousinjection of LPS. As a first application of the method for the detectionof cytokine induction in vivo, serial blood samples from healthyvolunteers injected with a low dose (4 ng/kg) of bacteriallipopolysaccharide was analysed. A clear induction of both IL-1RA andIL-1β mRNA was observed (FIG. 38). The induction of IL-1β mRNA wasrapid, since it was already detected 30 to 60 minutes after endotoxinadministration, and transient as IL-1β mRNA levels returned topre-injection values after 6 hours. IL-1 RA mRNA was also induced, witha delayed kinetics as compared to IL-1β mRNA.

Detection of anti-tetanus toxoid immune response after recallvaccination. As the in vitro experiments suggested that IL-2 mRNA wasthe most sensitive parameter to monitor anti-tetanus toxoid responses,this parameter was chosen to analyse the changes in the T cell responsesto tetanus toxoid in whole blood upon recall vaccination in vivo. Forthis purpose, whole blood incubation in absence or presence of tetanustoxoid was performed before and at several time points afteradministration of the vaccine. As shown in FIG. 39, the production ofIL-2 mRNA in whole blood exposed to the antigen significantly increasedin all vaccinated individuals. IL-2 mRNA induction was already apparent7 days post vaccination, maximal levels being reached at day 14 or 21.The variability between individuals is probably related to differencesin the basal status of anti-tetanus immunity (see also Table 3). TheIL-2 response measured in whole blood after vaccination was specific forthe immunising antigen as IL-2 mRNA levels measured in absence of invitro restimulation were not significantly modified (Table 3).

Discussion

Real time PCR is so called because the amplicon accumulation can bedirectly monitored during the PCR process, using fluorogenic moleculesthat bind the PCR product. This leads to the generation of afluorescence curve for each sample, from which it is possible todetermine the (c)DNA copy number of the sample, by comparison tofluorescence curves obtained with calibrated standards. In order toenhance the specificity, the fluorogenic molecule can be anoligonucleotide complementary to a sequence of the PCR product,localised between the two primers. The new methodology, as described inthe present application, provides a sensitive and accurate way toquantify nucleic acids in biological samples which was not possibleusing the prior art methods. The present application illustrates this byquantifying cytokine mRNA from purified cells or tissues representativeof the in vivo situation.

One of the difficulties encountered using whole blood for RT-PCRanalysis is the cell lysis that precedes RNA extraction. Because of thehigh amount of proteins present in plasma and erythrocytes, the majorityof the methods that isolate RNA from whole blood involve thepurification of the potential cellular sources of the analysed mRNA orthe elimination of the red blood cells, before performing the RNAextraction. These intermediate steps can be associated with mRNAdegradation and/or gene induction and thus with changes in mRNA levels.Furthermore, the simple fact of taking blood can lead to degradation ofsome mRNAs. This is especially true for cytokine mRNAs, which aresensitive to endogenous nucleases via the AU-rich sequences located intheir 3′ untranslated region. It was previously shown that peripheralblood IFN-γ mRNA levels indeed decreased by roughly 50% already one hourafter blood collection (Stordeur et al., (2002) J. Immunol. Meth.261:195). This can be avoided using quaternary amine surfactants such astetradecyltrimethylammonium oxalate, a cationic surfactant calledCatrimox-14™ (Qiagen, Westburg, Leusden, The Netherlands) that induceswhole cell lysis and, in the same time, nucleic acid precipitation. Thepresent example observes that the nucleic acid precipitate obtained withthe PAXgene™ tubes can surprisingly be dissolved in aguanidium/thiocyanate solution. An example of said solution is the lysisbuffer provided with the MagNA Pure™ LC kits for mRNA isolation (RocheApplied Science). This prompted us to combine the use of PAXgene™ tubeswith the MagNA Pure™ instrument, taking advantage of the highreproducibility and accuracy of the latter device due to the automatedpreparation of all of the components of the PCR reaction mixture.

Interestingly, the method of the present application was successfullyapplied to the detection of cytokine gene induction in whole blood uponendotoxin challenge in vivo, demonstrating that it could be used tomonitor systemic inflammatory responses. The transient nature of theIL-1 response after in vivo challenge, contrasts with the persistentincrease in IL-1 mRNA after in vitro addition of LPS to blood. Thismight be related to the rapid clearance of LPS in vivo but also to theredistribution of cytokine-producing cells in vivo, which is related toupregulation of adhesion molecules and chemokine receptors. Anotherpossible application of this whole blood method is the monitoring of Tcell responses upon vaccination, as suggested by the clear induction ofIL-2 mRNA observed after in vitro rechallenge in individuals vaccinatedwith tetanus toxoid. This might be of special interest for large-scalevaccination studies in which cell isolation might be difficult toorganise in good conditions, especially in developing countries whereseveral new vaccines are under evaluation. To further investigate theapplicability of this method in vaccine trials, it will be soon testedas read-out of T cell responses upon primary vaccination againsthepatitis B.

The direct correlation between the starting volume of blood and the mRNAcopy numbers (FIG. 36) suggests that there is no absolute need tomeasure mRNA concentration for expression of the results using thismethod. However, because even small variations of the sample volumecould result in quantification errors, it is preferable to correct themeasured copies by simultaneous measurement of a housekeeping gene suchas β-actin. This might still not be optimal as the expression ofhousekeeping genes might vary in certain conditions of stimulation.Therefore an external standard could be added to the sample before mRNAextraction. When the cellular source of a cytokine is well establishedsuch as in the case of T cells for IL-2, it might be appropriate tocorrect the numbers of cytokine gene copies by the numbers of copiesencoding a gene specifically expressed in the corresponding cell type,such as CD3 in the latter example. Likewise, internationalstandardisation of calibrators for cytokine mRNA quantification by realtime PCR should be developed to facilitate comparison of data generatedin different laboratories. Cytokine mRNA measurement in whole blood isuseful for the monitoring of innate and adaptive immune responsesrequired for the assessment of new vaccines and immunotherapies.

Example 7 Automated mRNA Extraction and Reagent Mix Preparation on theMagNA Pure: Direct Correlation Between Amount of Starting BiologicalMaterial and Found Copy Number

The procedure followed in this example is summarized in FIG. 40. Inorder to illustrate the accuracy of the system, a linear regression ofmRNA copy number on starting cell number was calculated (FIG. 41). mRNAwas extracted from various peripheral blood mononuclear cell (PBMC)numbers (ranging from 100,000 to 600,000 cells, X-axis) and one stepRT-real time PCR for β-actin mRNA was performed as described in the“Material and Methods” section of the present example 6. This experimenthas been repeated from PBMC for β-actin and TNF-α mRNAs (FIG. 42, panelsB and D), and from whole blood (FIG. 42, panel A) and CD4⁺ purified Tcells (FIG. 42, panel C) for β-actin mRNA.

Example 8 Cancer Immunotherapy

The procedure followed in this example is summarized in FIG. 40. Themethodology was applied to the monitoring of immune response induced bycancer vaccine. FIGS. 43, 40 and 41 illustrate the results obtained inthis field with a melanoma patient.

Example 9 Allergy

The procedure followed in this example is summarized in FIG. 40. Themethodology was then applied in Allergy. The response induced by invitro incubation of whole blood of an allergic subject with the relevantallergen was analysed by IL-4 mRNA quantification using real-time PCR.FIGS. 46, 47, 48 and 49 illustrate the results obtained in this field.

Example 10 Autoimmunity

The procedure followed in this example is summarized in FIG. 40. Themethodology was then applied in Autoimmunity. IL-2 mRNA quantificationusing this whole blood system was applied to assess T cell response toglutamic acid decarboxylase 65 (GAD65), an autoantigen being the targetof auto-reactive T cells in type 1 autoimmune diabetes. FIGS. 40 and 41illustrate the results obtained in this field.

Example 11 Transplantation

The procedure followed in this example is summarized in FIG. 40. Themethodology was then applied in Transplantation. IL-2 mRNAquantification by real time PCR after whole blood incubation withalloreactive non-T cells provides an alternative to the classical mixedlymphocytes reaction (MLR) to monitor alloreactive T cell response.FIGS. 52 and 53 illustrate the results obtained in this field.

It is to be understood that while the invention has been described inconjunction with the detailed description thereof, the foregoingdescription is intended to illustrate and not limit the scope of theinvention, which is defined by the scope of the appended claims. Otheraspects, advantages, and modifications are within the scope of thefollowing claims.

TABLE 1 Comparison of Qiagen and MagN A Pure LC extraction methods.IFN-gamma mRNA copy numbers per million of beta-actin mRNA copies 1.1.Qiagen mRNA extraction method. Blood mRNA coming from the same bloodsample was extracted 9 times. result 1 35 result 2 25 result 3 29 result4 27 result 5 27 result 6 49 result 7 33 result 8 22 result 9 27 mean 30SD 8 CV 26 1.2. MagNA Pure LC (kit + instrument) mRNA extraction method.Blood mRNA prepared from the same blood sample was extracted 9 times.result 1 192 result 2 170 result 3 153 result 4 139 result 5 138 result6 160 result 7 105 result 8 142 result 9 142 mean 149 SD 24 CV 16

TABLE 2 Oligonucleotides for (real time) PCR¹PRIMERS AND PROBES FOR REAL TIME PCR Final Product concen- mRNA sizetration target Oligonucleotides (5′→3′)² (bp) (nM)³ IL-2F273: CTCACCAGGATGCTCACATTTA 95 F 900 R367: TCCAGAGGTTTGAGTTCTTCTTCTR 900 P304: 6Fam-TGCCCAAGAAGGCCACAGAACTG-Tamra-p IL-4F174: ACTTTGAACAGCCTCACAGAG 74 F 300 R247: TTGGAGGCAGCAAAGATGTC R 900P204: 6Fam-CTGTGCACCGAGTTGACCGTA-Tamra-pPRIMERS FOR STANDARD PREPARATION BY “CLASSICAL” PCR⁴ mRNA Product targetOligonucleotides (5′→3′)² size (bp) IL-2 F155: TGTCACAAACAGTGCACCTACT518 R672: AGTTACAATAGGTAGCAAACCATACA IL-4 F27: TAATTGCCTCACATTGTCACT 503R529: ATTCAGCTCGAACACTTTGAA ¹For a full description, see Stordeur et al,J Immunol Methods, 259 (1-2): 55-64, 2002. ²F, R and P indicate forwardand reverse primers and probes, respectively; numbers indicate thesequence position from Genebank accession numbers X01586 for IL-2 andNM_000589 for IL-4. ³Final concentration of forward (F) and reverse (R)primers. ⁴Standard curves were generated from serial dilutions of PCRproducts prepared by “classical” PCR, for which specific conditions wereas follows: denaturation at 95° C. for 20 s,. annealing at 58° C. for 20s and elongation at 72° C. for 45 s, for a total of 35 cycles. MgCl2final concentration was 1.5 mM.

TABLE 3 Tetanus Adult whole blood Toxoid Cord blood (before vaccinerecall) −− 109 ± 51 1,154 ± 1,194 + 159 ± 91 7,715 ± 8,513

1. A kit for assaying a liquid biological sample comprising: a vesselsuitable for accepting liquid biological sample, exposing said sample toa first substance and subsequently a nucleic acid stabilising agent,said vessel comprising: a) a first substance present inside said vessel,b) a container in which said stabilising agent is present, c) aconnection between the inside of said vessel and the inside of saidcontainer, d) a physical barrier that temporarily blocks saidconnection; and a control vessel suitable for accepting liquidbiological sample, exposing said sample to a control substance andsubsequently a nucleic acid stabilising agent, said control vesselcomprising: a) a control substance present inside said control vessel,b) a control container in which said stabilising agent is present, c) aconnection between the inside of said control vessel and the inside ofsaid control container, d) a physical barrier that temporarily blockssaid connection.
 2. The kit according to claim 1, wherein said firstsubstance is immobilised on part or all of the inside surface of saidvessel.
 3. The kit according to claim 1, wherein said first substance isimmobilised on a solid support.
 4. The kit according to claim 1, whereinsaid first substance is a liquid.
 5. The kit according to claim 1,wherein said first substance is a solid.
 6. The kit according to claim1, wherein the vessel and/or control vessel comprise one or more areassuitable for puncture by a syringe needle.
 7. The kit according to claim6, wherein said area is a re-sealable septum.
 8. The kit according toclaim 1, wherein the vessel and/or control vessel comprise a fittingsuitable for receiving a syringe and transmitting the contents thereinto the interior of said vessel or control vessel.
 9. The kit accordingto claim 1, wherein the vessel and/or control vessel comprise a fittingsuitable for receiving a syringe needle.
 10. The kit according to claim1, wherein the vessel and/or control vessel comprise a valve which iscapable of minimising the flow of gas/liquid from vessel, and allowingthe flow of liquid biological sample into the vessel.
 11. The kitaccording to claim 1, wherein the vessel and/or control vessel comprisea means through which displaced gas may be expelled.
 12. The kitaccording to claim 1, wherein the vessel and/or control vessel are heldunder negative pressure.
 13. The kit according to claim 1, wherein thephysical barrier of item d) is opened by the application of physicalforce to said vessel or control vessel.
 14. The kit according claim 13,wherein said force transmits an opening means to said physical barrier.15. The kit according to claim 13 wherein said force irreversibly openssaid physical barrier.
 16. The kit according to claim 1, wherein saidvessel and/or control vessel comprise an indication for dispensing aknown volume of stabilising agent therein.
 17. The kit according toclaim 1, wherein said first substance comprises one or more immunesystem antigens.
 18. The kit according to claim 17 wherein said immunesystem antigens are vaccine components.
 19. The kit according to claim17 wherein said immune system antigens are antigens which provoke ahyperallergenic response.
 20. The kit according to claim 17 wherein saidimmune system antigens are one or more selected from histocompatibilityantigens, bacterial LPS, tetanus toxoid, a cancer immunotherapy antigen,MAGE-3, a cat allergen, Feld1, antigen presenting cells from an organdonor, an autoantigen, and GAD65.
 21. The kit according to claim 1,wherein said stabilising agent is an inhibitor of cellular RNAdegradation and/or gene induction.
 22. The kit according to claim 1,wherein the exterior of the vessel and control vessel are joined to forma single entity.